Nucleic Acid Synthesizers

ABSTRACT

The present invention relates to nucleic acid synthesizers and methods of using and modifying nucleic acid synthesizers. For example, the present invention provides highly efficient, reliable, and safe synthesizers that find use, for example, in high throughput and automated nucleic acid synthesis, as well as methods of modifying pre-existing synthesizers to improve efficiency, reliability, and safety.

This application is a Continuation of application Ser. No. 10/054,023,filed on Nov. 13, 2001, which issued on Oct. 14, 2008 as U.S. Pat. No.7,435,390 and which is incorporated herein by reference, which is aContinuation-in-Part of application Ser. No. 10/002,251, filed Oct. 26,2001, now abandoned, which is a Continuation-in-Part of application Ser.No. 09/782,702, filed Feb. 13, 2001, now abandoned, which is aContinuation-in-Part of application Ser. No. 09/771,332, filed Jan. 26,2001, which issued on Aug. 23, 2005 as U.S. Pat. No. 6,932,943.

FIELD OF THE INVENTION

The present invention relates to nucleic acid synthesizers and methodsof using and modifying nucleic acid synthesizers. For example, thepresent invention provides highly efficient, reliable, and safesynthesizers that find use, for example, in high throughput andautomated nucleic acid synthesis, as well as methods of modifyingpre-existing synthesizers to improve efficiency, reliability, andsafety. The present invention also relates to synthesizer arrays forefficient, safe, and automated processes for the production of largequantities of oligonucleotides.

BACKGROUND

With the completion of the Human Genome Project and the increasingvolume of genetic sequence information available, genomics research andsubsequent drug design efforts have been increasing as well. Manydiagnostic assays and therapeutic methods utilize oligonucleotides. Theinformation obtained from genomic analysis provides valuable insightinto the causes and mechanisms of a large variety of diseases andconditions, while oligonucleotides can be used to alter gene expressionin cells and tissues to prevent or attenuate diseases or alterphysiology. As more nucleic acid sequences continue to be identified,the need for larger quantities of oligonucleotides used in assays andtherapeutic methods increases.

To meet the increasing demand for nucleic acid synthesis, there has beenan increase in the variety of designs, and the volume of production ofnucleic acid synthesizers. Unfortunately, the currently availablesynthesizers are not designed to adequately meet the needs of theindustry. Exemplary, nucleic acid synthesizers include the synthesizersdescribed in US Patent Publication No. 2001/0001035 A1, published on May10, 2001, and incorporated herein in its entirety for all purposes. Yet,this type of synthesizer has a significant number of drawbacks. Inparticular, available synthesizers are limited in their ability toefficiently synthesize large numbers of oligonucleotides. Whilesynthesizers have been developed to simultaneously synthesize more thanone oligonucleotide at a time, such machines are not efficient at theproduction of different types of nucleic acids simultaneously (e.g.,different lengths of nucleic acids) and are unacceptably prone toperformance failures and environmental contamination. Furthermore,available synthesizers are not suitably configured for use inlarge-scale nucleic acid production facilities or for automated nucleicacid synthesis. Thus, the art is in need of nucleic acid synthesizersthat are safe, efficient, flexible, and are amenable to large-scaleproduction and automation.

SUMMARY OF THE INVENTION

While the present invention will be described with reference to severalspecific embodiments, the description is illustrative of the presentinvention and is not to be construed as limiting the invention. Variousmodifications to the present invention can be made without departingfrom the scope and spirit of the present invention. For the sake ofclarity and a better understanding of the present invention, commoncomponents share common reference numerals throughout various figures.

The present invention relates to nucleic acid synthesizers and methodsof using and modifying nucleic acid synthesizers. For example, thepresent invention provides highly efficient, reliable, and safesynthesizers that find use, for example, in high throughput andautomated nucleic acid synthesis, as well as methods of modifyingpre-existing synthesizers to improve efficiency, reliability, andsafety. The present invention also relates to synthesizer arrays forefficient, safe, and automated processes for the production of largequantities of oligonucleotides.

In some embodiments, the present invention provides systems comprising asynthesis and purge component, the synthesis and purge componentcomprising a cartridge and a drain plate, wherein the cartridge isconfigured to hold one or more nucleic acid synthesis columns andwherein the cartridge is separated from the drain plate by a drain plategasket. In certain embodiments, the cartridge is configured to hold aplurality of nucleic acid synthesis columns. In particular embodiments,the cartridge is configured to hold 12 or more nucleic acid synthesiscolumns. In other embodiments, the cartridge is configured to hold 48 ormore nucleic acid synthesis columns. In additional embodiments, thecartridge is configured to hold exactly 48 nucleic acid synthesiscolumns.

In some embodiments, the assembly comprising the cartridge, the drainplate and the drain plate gasket is configured to provide asubstantially airtight seal between the assembly and the outside of eachnucleic acid synthesis column. In one embodiment, the airtight sealbetween the assembly and each column is provided by an O-ring. In apreferred embodiment, each O-ring is positioned between the cartridgeand the exterior surface of a column.

In certain embodiments, the drain plate gasket provides a substantiallyairtight seal between the cartridge and the drain plate. In otherembodiments, the drain plate gasket provides an airtight seal betweenthe cartridge and the drain plate. In some embodiments, the drain plategasket comprises one or more alignment markers configured to allowaligned attachment of said cartridge to said drain plate. In additionalembodiments, the drain plate gasket comprises one or more alignmentmarkers configured to allow aligned attachment of the drain plate gasketto the cartridge. In other embodiments, the drain plate gasket comprisesone or more alignment markers configured to allow aligned attachment ofthe gasket to the drain plate. In certain embodiments, the drain plategasket comprises at least one drain cut-out. In other embodiments, thedrain plate gasket comprises at least four drain cut-outs. In stillother embodiments, the drain plate gasket comprises one drain cut outfor every synthesis column in the cartridge. In yet other embodiments,the cut outs in the drain plate gasket for each synthesis column areconfigured to provide an airtight seal between the outside of eachnucleic acid synthesis column and the assembly comprising the cartridge,the drain plate, and the drain plate gasket.

In some embodiments, the present invention provides systems comprising asynthesis and purge component, the synthesis and purge componentcomprising a cartridge and a drain plate, wherein the cartridge isconfigured to hold one or more nucleic acid synthesis columns andwherein the cartridge is separated from the drain plate by a drain plategasket. In some embodiments, the drain plate comprises at least onedrain (e.g. 1, 2, 3, 4, 5, 10, . . . 20, . . . ). In other embodiments,the system further comprises a waste tube 63, the waste tube comprisinginput and output ends, wherein the input end is configured to receivewaste materials from the drain. In particular embodiments, the wastetube comprises an inner diameter of at least 0.187 inches (preferably atleast 0.25 inches). In some embodiments, the waste tube and the drainare configured such that, when the drain is contacted with the wastetube for waste removal, the waste tube encloses at least a portion ofthe drain (See, e.g., FIG. 8). In particular embodiments, the drainforms a sealed contact point with an interior portion of the waste tubewhen the drain is enclosed in the waste tube. In still otherembodiments, the drain further comprises a drain sealing ring. Incertain embodiments, the system further comprises a waste valve whereinthe waste valve is configured to receive waste from the output end ofthe waste tube. In particular embodiments, the waste valve comprises aninterior diameter of at least 0.187 inches (preferably at least 0.25inches). In some embodiments, the waste valve provides astraight-through path for the waste (e.g. as opposed to an angled path).Straight-through paths can be accomplished, for example, by the use of agate or ball valve.

In some embodiments, the system further comprises a plurality ofdispense lines, the dispense line configured for delivering at least onereagent to a synthesis column in the cartridge. In certain embodiments,the dispense lines comprise an interior diameter of at least 0.25 mm. Inparticular embodiments, the system further comprises an alignmentdetector. In particular embodiments, the alignment detector isconfigured to detect the alignment of a waste tube and a drain. In otherembodiments, the alignment detector is configured to detect thealignment of a dispense line and a receiving hole of the cartridge. Insome embodiments, the alignment detector is configured to detect a tiltalignment of the synthesis and purge component.

In some embodiments, the system of the present invention furthercomprises a motor attached to the synthesis and purge component andconfigured to rotate the synthesis and purge component. In particularembodiments, the motor is attached to the synthesis and purge componentby a motor connector. In further embodiments, the system furthercomprises a bottom chamber seal positioned between the motor connectorand the synthesis and purge component. In certain embodiments, thesystem of the present invention comprises two drain. In preferredembodiments, the two drain are located on opposite sides of the drainplate.

In some embodiments of the systems of the present invention, thesynthesis and purge component is contained in a chamber. In certainembodiments, a chamber bowl and a top cover (when in place) combine toform a chamber (e.g. which may be pressurized, for example, with inertgas). One example is depicted in FIG. 2 where chamber bowl 18 and topcover 30 combine to form an exemplary chamber. In some embodiments, thechamber comprises a bottom surface (e.g. bottom of a chamber bowl, see,e.g. FIG. 9) comprising the top portion of two waste tubes (which may,for example, extend downward from bottom of the chamber). In preferredembodiments, the waste tubes are positioned symmetrically on the bottomsurface of the chamber (see, e.g., FIG. 9).

In particular embodiments, the systems of the present invention furthercomprise a chamber drain having open and closed positions, the chamberdrain configured to allow gas emissions (or liquid waste) to pass out ofthe chamber when in the open position.

In some embodiments, the systems of the present invention furthercomprise a reagent dispensing station, wherein the reagent dispensingstation is configured to house one or more reagent reservoirs, such thatreagents in reagent reservoirs can be delivered to the cartridge. Incertain embodiments, the reagent dispensing station comprises one ormore ventilation tubes (e.g., connected to one or more ventilationvalves of the reagent dispensing station) configured to remove gaseousemissions from the reagent dispensing station. In certain embodiments,the reagent dispensing station provides an enclosure. In preferredembodiments, the enclosure comprises a viewing window to allow visualinspection of the reagent reservoirs without opening the enclosure. Inpreferred embodiments, one reagent dispensing station is configured toserve multiple synthesizers.

In some embodiments, the systems of the present invention comprise anintegrated ventilation system, e.g., a fume hood, wherein the fume hoodis configured to draw gaseous emissions away from an instrumentoperator. In some embodiments, the integrated fume hood comprises sidepanels and a front panel, wherein said side and front panels create aventilated workspace having negative air pressure when compared with theambient environment. In preferred embodiments, the side and front panelscreate the ventilated workspace when the synthesizer is opened, e.g.,for operator access to the reaction enclosure. In some embodiments, thepanels fold or slide into recesses in the synthesizer body upon closingof the instrument, such that the instrument can be closed withoutremoval of the panels.

In particular embodiments, the systems of the present invention arecapable of maintaining a gas pressure in the chamber sufficient to purgesynthesis columns prior to addition of reagents to the synthesiscolumns.

In some embodiments, the nucleic acid synthesis systems of the presentinvention comprise a cartridge in a chamber, the cartridge comprising aplurality of synthesis columns, wherein the synthesis columns containpacking material that provides a resistance against pressurized gascontained in the chamber, the resistance being sufficient to maintain apressure in the chamber that is capable of purging synthesis columnsprior to addition of reagents to the synthesis columns. In certainembodiments, one or more of the plurality of synthesis columns does notundergo a synthesis reaction. In particular embodiments, two or moredifferent lengths of oligonucleotides are synthesized in the pluralityof synthesis columns. In other embodiments, the packing materialcomprises a frit. In some embodiments, the frit is a bottom frit. Inother embodiments, the frit is a top frit. In preferred embodiments, thepacking material comprises a top frit, solid support, and a bottom frit.In particularly preferred embodiments, the solid support is polystyrene.In some embodiments, the packing material comprises a synthesis matrix.

In some embodiments, the present invention provides nucleic acidsynthesis systems comprising a synthesis and purge component in apressurized chamber, the synthesis and purge component comprising aplurality of synthesis columns, wherein the synthesis columns containpacking material sufficient to maintain pressure in the chamber during apurging operation to purge liquid reagent from the plurality ofsynthesis columns when at least one of the plurality of synthesiscolumns does not contain liquid reagent. In certain embodiments, morethan one of the plurality of synthesis columns (e.g. 2, 3, 5, 10) do notcontain liquid reagent (and the remaining synthesis columns do containliquid reagent).

In certain embodiments, the present invention provides nucleic acidsynthesis systems comprising: a) a synthesis and purge component, thesynthesis and purge component comprising a cartridge and a drain plateseparated by a drain plate gasket, wherein the cartridge is configuredto hold twelve or more nucleic acid synthesis columns; b) a drainpositioned in the drain plate; c) a chamber comprising an inner surface,the chamber housing the synthesis and purge component and the drain; d)a waste tube, the waste tube comprising input and output ends, whereinthe input end is configured to receive waste materials from the drain,wherein the waste tube comprises an inner diameter of at least 0.187inches; e) a waste valve configured to receive waste from the output endof the waste tube, wherein the waste valve comprises in interiordiameter of at least 0.187 inches; f) a reagent dispensing station,wherein the reagent dispensing station is configured to house one ormore reagent reservoirs; g) a plurality of dispense lines, the dispenselines configured for delivering reagents from the reagent reservoirs toa synthesis column in the cartridge, wherein the dispense lines comprisean interior diameter of at least 0.25 mm) a rotating motor attached tothe synthesis and purge component by a motor connector and configured torotate the synthesis and purge component; and i) a gas line configuredto release gas into the chamber to create a gas pressure in the chambergreater than a gas pressure in the waste tube. In certain embodiments,the system is capable of maintaining gas pressure in the chamber at asufficient level to purge the synthesis columns prior to addition ofreagents to the synthesis columns.

In some embodiments, the synthesizer further comprises providing energy,such as heat, to the synthesis columns. Heating of the synthesis columnfinds use, for example, in decreasing the coupling time during a nucleicacid synthesis. It can also broaden the range of the chemical protocolsthat can be used in high throughput synthesis, e.g. by improving theefficiency of less efficient chemistries, such as the phosphate triestermethod of oligonucleotide synthesis. In other embodiments, thesynthesizer further comprises a mixing component, such as an agitator,configured to agitate the synthesis columns (e.g., to mix reactioncomponents, and to facilitate mass exchange between the reaction mediumand the solid support).

In some embodiments, the present invention provides methods forsynthesizing nucleic acids comprising: a) providing: i) a nucleic acidsynthesizer comprising a synthesis and purge component, the synthesisand purge component comprising a cartridge and a drain plate, whereinthe cartridge holds a plurality of nucleic acid synthesis columns andwherein the cartridge is separated by a drain plate gasket from thedrain plate, and ii) nucleic acid synthesis reagents; and b) introducinga portion of the nucleic acid synthesis reagents into at least one ofthe nucleic acid synthesis columns to provide a first synthesisreaction; c) purging the nucleic acid synthesis columns by creating apressure differential across the nucleic acid synthesis columns; and d)introducing a second portion of the nucleic acid synthesis reagents intoat least one of the nucleic acid synthesis columns to provide a secondsynthesis reaction. In particular embodiments, the drain plate gasketprovides a substantially airtight seal between the cartridge and thedrain plate. In other embodiments, the drain plate gasket provides anairtight seal between the cartridge and the drain plate.

The present invention further provides a cartridge for use in an opennucleic acid synthesis system, said cartridge comprising a plurality ofreceiving holes configured to hold nucleic acid synthesis columns,wherein the cartridge is further configured to receive one or moreO-rings, wherein the presence of the one or more O-rings provides a sealbetween the nucleic acid synthesis columns and the plurality ofreceiving holes (i.e., the O-ring contacts an interior wall of thereceiving hole and an exterior wall of the synthesis column to form aseal). In some embodiments, the cartridge is provided as part of anucleic acid synthesis system. The present invention is not limited bythe nature of the O-ring. For example, in some embodiments, thecartridge is associated with a gasket, wherein the gasket provides theO-rings (e.g., through one or more holes in the gaskets, such that whenthe gasket is associated with the cartridge [e.g., affixed to an outersurface of the cartridge] a seal is formed between the a receiving holeof the cartridge and a synthesis column within the receiving hole [seee.g., FIG. 12C]). In other embodiments, the O-ring is provided in agroove within the receiving hole. For example, in some embodiments, thegroove is located at the top surface of the receiving hole. In suchembodiments, the plurality of receiving holes comprise an upper portionand a lower portion, wherein the lower portion comprises a firstdiameter and the upper portion comprises a second diameter that islarger than the first diameter (see e.g., FIG. 12A). In otherembodiments, the groove is located within an interior portion of thereceiving hole. In such embodiments, the plurality of receiving holescomprise an upper portion with a first diameter, a middle portion with asecond diameter, and a lower portion with a third diameter, wherein thesecond diameter is larger than the first diameter and larger than thethird diameter (the first and third diameters may be the same as eachother or different). When an O-ring is placed in the groove, the O-ringcontains an internal diameter less than the first diameter and less thanthe third diameter, such that it can contact a synthesis column placedwithin the receiving hole (see e.g., FIG. 12B).

In some embodiments, the cartridge comprises a rotary cartridge. In somepreferred embodiments, O-rings are provided in the cartridge. In somepreferred embodiments, the O-ring is configured to form a substantiallyairtight or pressure-tight seal between the receiving hole and thenucleic acid synthesis column, when said nucleic acid synthesis columnis present.

The present invention further provides a nucleic acid synthesis systemcomprising a synthesis and purge component in a pressurizable chamber,said synthesis and purge component comprising a cartridge, wherein thecartridge in configured to hold a plurality of nucleic acid synthesiscolumns, and wherein said cartridge is further configured to providesseals between said cartridge and each of said plurality of nucleic acidsynthesis columns so as to maintain pressure in said chamber during apurging operation to purge liquid reagent from said plurality ofsynthesis columns. In some embodiments, each of the seals between thecartridge and the plurality of nucleic acid synthesis columns isprovided by an O-ring.

In some embodiments, the present invention provides a nucleic acidsynthesizer comprising a plurality of synthesis columns and an energyinput component that imparts energy to said plurality of synthesiscolumns to increase nucleic acid synthesis reaction rate in saidplurality of synthesis columns. In some embodiments, said energy inputcomponent comprises a heating component. In preferred embodiments, saidheating component provides substantially uniform heat. In someembodiments, said energy input component provides heated reagentsolutions to said plurality of synthesis columns. In other embodiments,said energy input component comprises a heating coil. In yet otherembodiments, said energy input component comprises a heat blanket. Inyet other embodiments, said heating component comprises a resistanceheater, a Peltier device, a magnetic induction device or a microwavedevice. In still other embodiments, said energy input componentcomprises a heated room. In further embodiments, said energy inputcomponent provides energy in the electromagnetic spectrum. In yet otherembodiments, said energy input component comprises an oscillatingmember. In some embodiments, said energy input component provides aperiodic energy input, and in other embodiments, said energy inputcomponent provides a constant energy input.

In some preferred embodiments, said energy input heats said plurality ofsynthesis columns in the range of about 20 to about 60 degrees Celsius.

In some embodiments, the present invention provides a nucleic acidsynthesizer comprising a fail-safe reagent delivery component configuredto deliver one or more reagent solutions to said plurality of synthesiscolumns. In some embodiments, the fail-safe reagent delivery componentcomprises a plurality of reagent tanks. In preferred embodiments, saidplurality of reagent tanks comprise one or more tanks selected from thegroup consisting of acetonitrile tanks, phosphoramidite tanks, argon gastanks, oxidizer tanks, tetrazole tanks, and capping solution tanks. Insome particularly preferred embodiments, said reagent tanks comprise aplurality of large volume containers, each said large volume containercomprising at least one of said reagent solutions. In some embodiments,the present invention provides high-throughput oligonucleotideproduction systems comprising: an oligonucleotide synthesizer array,wherein the oligonucleotide synthesizer array comprises at least 5oligonucleotide synthesizers. In preferred embodiments, theoligonucleotide synthesizer array comprises at least 10 or at least 100oligonucleotide synthesizers. In certain embodiments, the system furthercomprises a centralized control network operably linked to theoligonucleotide synthesizer component.

In particular embodiments, the present invention provides methods forthe high through-put production of oligonucleotides comprising; a)providing an oligonucleotide synthesizer array; and b) generating a highthrough-put quantity of oligonucleotides with the oligonucleotidesynthesizer array, wherein the high through-put quantity comprises atleast 1 per hour (e.g. at least 1, 10, 100, 1000, etc, per hour).

The present invention provides a production facility comprising an arrayof synthesizers. In some embodiments, the production facility of thepresent invention comprises a fail-safe reagent delivery system. Inother embodiments, the production facility of the present inventioncomprises a centralized waste collection system. In yet otherembodiments, the production facility of the present invention comprisesa centralized control system. In preferred embodiments, the productionfacility of the present invention comprises a fail-safe reagent deliverysystem, a centralized waste collection system and a centralized controlsystem.

In some embodiments, the present invention provides an automatedproduction process. In some embodiments, the automated productionprocess includes an oligonucleotide synthesizer component and anoligonucleotide-processing component.

The present invention also provides integrated systems that link nucleicacid synthesizers to other nucleic acid production components. Forexample, the present invention provides a system comprising a nucleicacid synthesizer and a cleavage and deprotect component. In someembodiments, the synthesizer is configured for parallel synthesis ofnucleic acid molecules in three or more synthesis columns. In someembodiments, the system further comprises sample tracking softwareconfigured to associate sample identification tags (e.g., electronicidentification numbers, barcodes) with samples that are processed by thenucleic acid synthesizer and the cleavage and deprotect component. Insome preferred embodiments, the sample tracking software is furtherconfigured to receive synthesis request information from a user, priorto sample processing by the nucleic acid synthesizer. In someembodiments, the system further comprises a robotic component configuredto transfer columns from the nucleic acid synthesizer to the cleavageand deprotect component. In other preferred embodiments, the roboticcomponent is further configured to transfer the columns from thecleavage and deprotect component to a purification component and/or toadditional production components described herein.

The present invention also provides control systems for operating one ormore components of the systems of the present invention. For example,the present invention provides a system comprising a processor, whereinthe processor is configured to operate a nucleic acid synthesizer forparallel synthesis of three or more nucleic acid molecules.

The present invention further provides a system comprising a processor,wherein said processor is configured to operate a nucleic synthesizerand a cleavage and deprotect component. In some embodiments, the systemfurther comprises a computer memory, wherein the computer memorycomprises nucleic acid sample order information (e.g., informationobtained from a user specifying the identity of a polymer to besynthesized and/or specifying one or more characteristics of the polymersuch as sequence information). In some embodiments, the computer memoryfurther comprises allele frequency information and/or diseaseassociation information.

In some embodiments, the present invention provides oligonucleotidesynthesizers comprising a reaction chamber and a lid, wherein in an openposition, the lid provides a substantially enclosed ventilatedworkspace. In certain embodiments, the present invention providesmethods of protecting an operator of an oligonucleotide synthesizercomprising channeling ambient air away from an operator toward aninterior space of the synthesizer (e.g. down through the top surface, orup through the top cover). In other embodiments, the present inventionprovides apparatuses comprising, in combination, an oligonucleotidesynthesizer and a venting hood. In some embodiments, the apparatuses arefor production of oligonucleotides, wherein the apparatus comprises aventing component configured to draw air away from a reaction chamber ofthe apparatus. In certain embodiments, the present invention providessystems comprises a plurality of oligonucleotide apparatuses (e.g. e.g.at least 100 synthesizers).

In particular embodiments, the present invention provides a polymersynthesizer comprising a ventilated workspace. In some embodiments,certain embodiments, the polymer synthesizer is a nucleic acidsynthesizer. In certain embodiments, the synthesizer comprises a topenclosure, wherein the top enclosure comprises a top plate with aventilation opening, wherein the top enclosure is configured forattachment to a top cover of a synthesizer to form a primarily enclosedspace over the top cover. In other embodiments, the synthesizercomprises a base, wherein the base comprises a primarily enclosed spaceand a ventilation opening.

In certain embodiments, the top plate is configured for attachment to aventilation tube such that air in the primarily enclosed space may bedrawn through the ventilation opening into the ventilation tube. Inother embodiments, the top plate further comprises an outer window, andwherein the ventilation opening is formed in the outer window. Incertain embodiments, the top enclosure further comprises at least foursides (e.g. 4 sides, 5 sides, etc.). In certain embodiments, the topcover further comprises a ventilation slot.

In certain embodiments, the present invention provides polymersynthesizer (e.g. nucleic acid synthesizer) comprising; a) a top coverwith a ventilation slot, and b) a top enclosure, wherein the topenclosure comprises a top plate with a ventilation opening, and whereinthe top enclosure is attached to the top cover to form a primarilyenclosed space above the top cover.

In certain embodiments, the present invention provides a lid enclosurecomprising; a) a top cover with a ventilation slot, and b) a topenclosure, wherein the top enclosure comprises a top plate with aventilation opening, and wherein the top enclosure is attached to thetop cover to form a primarily enclosed space over the top cover. Incertain embodiments, the top plate is configured for attachment to aventilation tube. In particular embodiments, the top plate is configuredfor attachment to a ventilation tube such that air in the primarilyenclosed space may be drawn through the ventilation opening into theventilation tube. In other embodiments, the top cover is configured toattach to a top surface of a nucleic acid synthesizer with a chamberbowl.

In some embodiments, the ventilation slot is configured such that air inthe chamber bowl may drawn in through the ventilation slot and into theprimarily enclosed space. In other embodiments, the top plate furthercomprises an outer window, and wherein the ventilation opening is formedin the outer window. In certain embodiments, the top enclosure furthercomprises at least four sides.

In certain embodiments, the present invention provides a polymersynthesizer (e.g., nucleic acid synthesizer) comprising; a) a topsurface of a nucleic acid synthesizer, b) a lid enclosure comprising; i)a top plate with a ventilation opening, and ii) a top cover with aventilation slot; and wherein the lid enclosure is attached to the topsurface. In some embodiments, the lid enclosure is attached to the topsurface by at least one hinge such that the lid enclosure may be raisedand lowered. In certain embodiments, the present invention providessystems comprises a plurality of the polymer synthesizers (e.g. e.g. atleast 100 synthesizers).

In some embodiments, the present invention provides side panelsconfigured to extend between at least one side of a top cover (or lidenclosure) and a top surface of a nucleic acid synthesizer such that abarrier to air is created on at least one side of the synthesizer whenthe top cover is extended upward from the top surface. In otherembodiments, the present invention provides a panel (e.g. front panel orside panel) configured to extend at least part way between at least oneside of a top cover (or lid enclosure) and a top surface of a nucleicacid synthesizer such that at least a partial barrier to air is createdon at least one side of the synthesizer when the top cover is extendedupward such that it is not in contact with the top surface. In otherembodiments, the present invention provides polymer synthesizers (e.g.nucleic acid synthesizers) summary comprising; a) a top surface of anucleic acid synthesizer, b) a lid enclosure comprising; i) a top platewith a ventilation opening, ii) a top cover with a ventilation slot; andiii) at least one top enclosure side; and c) a panel; wherein the lidenclosure is attached to the top surface by at least one hinge such thatthe lid enclosure may be raised and lowered, and wherein the panel isconfigured to extend (at least part way) between the at least one topenclosure side and the top surface such that at least a partial barrierto air is created when the lid enclosure is extended upward from the topsurface. In certain embodiments, the present invention provides systemscomprises a plurality of the polymer synthesizers (e.g. e.g. at least100 synthesizers).

In particular embodiments, the present invention provides systemscomprising; a) a ventilation tube, and b) a lid enclosure comprising; a)a top cover with a ventilation slot, and b) a top enclosure comprising atop plate with a ventilation opening, wherein the top enclosure isattached to the top cover to form a primarily enclosed space over thetop cover. In some embodiments, the systems further comprise a vacuumsource (e.g. centralized vacuum system).

In certain embodiments, the top plate is configured for attachment tothe ventilation tube. In other embodiments, the ventilation tube isconfigured for attachment to the vacuum source. In particularembodiments, the system further comprises a synthesis and purgecomponent, the synthesis and purge component comprising a cartridge anda drain plate separated by a drain plate gasket, wherein the cartridgeis configured to hold a plurality of nucleic acid synthesis columns. Insome embodiments, the systems further comprise a plurality of dispenselines, wherein the plurality of dispense lines are located in theprimarily enclosed space.

In certain embodiments, the systems further comprise at least one sidepanel, wherein the at least one side panel is configured to extendbetween at least one side of the lid enclosure and a top surface of anucleic acid synthesizer (e.g., such that a barrier to air is created onat least one side of the synthesizer when the top cover is extendedupward from the top surface).

In some embodiments, the present invention provides systems comprising;a) a nucleic acid synthesizer comprising; i) a top surface, and ii) atop cover comprising a ventilation slot, wherein the top cover isattached to the top surface by at least one hinge such that the topsurface may be raised and lowered; and b) a panel configured to extendat least part way between at least one side of the top cover and the topsurface such that at least a partial barrier to air is created on atleast one side of the nucleic acid synthesizer when the top cover isextended upward. In other embodiments, the panel is configured to fullyextend between the at least one side of the top cover and the topsurface such that a complete barrier to air is created on at least oneside of the nucleic acid synthesizer when the top cover is extendedupward. In some embodiments, the panel comprises a side panel or a frontpanel.

In certain embodiments, the system further comprises a top enclosure,wherein the top enclosure comprises a top plate with a ventilationopening, and wherein the top enclosure is attached to the top cover toform a primarily enclosed space over the top cover. In otherembodiments, the system further comprises a ventilation tube. Inparticular embodiments, the system further comprises a vacuum source. Inother embodiments, the vacuum source comprises a centralized vacuumsystem. In particular embodiments, the top plate is configured forattachment to the ventilation tube. In certain embodiments, theventilation tube is configured for attachment to the vacuum source.

In some embodiments, the present invention provides methods comprisingforming a ventilation opening in a top plate of a top enclosure suchthat the top plate is configured for attachment to a ventilation tube.In certain embodiments, the present invention provides methodscomprising; a) providing; i) a top enclosure comprising a top plate, andii) a ventilation tube; and b) forming a ventilation opening in the topplate, and c) attaching the ventilation tube to the top plate such thatthe ventilation tube forms a seal around the ventilation opening. Infurther embodiments, the methods further comprise step d) attaching aleast one panel to the top enclosure.

In other embodiments, the present invention provides methods comprising;a) providing; i) a top cover of a nucleic acid synthesizer comprising aventilation slot, wherein the top cover is configured to be attached toa top surface of a nucleic acid synthesizer such that the top surfacemay be raised and lowered; and ii) a top enclosure, wherein the topenclosure comprises a top plate with a ventilation opening, and b)attaching the top enclosure to the top cover such that a primarilyenclosed space is formed over the top cover. In other embodiments, themethods further comprise the step of attaching at least one panel to thetop enclosure (or the top cover), wherein the at least one panel extendsat least part way between at least one side of the top cover (or the topcover) and the top surface such that at least a partial barrier to airis created on at least one side of the synthesizer when the top cover isextended upward such that it is not in contact with the top surface.

In particular embodiments, the present invention provides methodscomprising; a) providing; i) a nucleic acid synthesizer comprising; i) atop cover with a ventilation slot, and ii) a top enclosure, wherein thetop enclosure comprises a top plate with a ventilation opening, whereinthe top enclosure is attached to the top cover to form a primarilyenclosed space above the top cover, and wherein the top plate isattached to a ventilation tube such that the ventilation tube forms aseal around the ventilation opening, and ii) a vacuum source attached tothe ventilation tube, and b) activating the vacuum source such that airis drawn into the ventilation slot, through the primarily open space,and out through the ventilation opening into the ventilation tube.

In some embodiments, the present invention provides kits comprising; a)a top enclosure comprising a top plate with a ventilation opening,wherein the top enclosure is configured for attachment to a top cover ofa synthesizer to form a primarily enclosed space over the top cover, andb) a printed material component, wherein the printed material componentcomprises written instruction for installing the top enclosure onto thetop cover.

In other embodiments, the present invention provides kits comprising; a)a panel configured to extend at least part way between at least one sideof a top cover (or lid enclosure) and a top surface of a nucleic acidsynthesizer such that at least a partial barrier to air is created on atleast one side of the synthesizer when the top cover is extended upwardsuch that it is not in contact with the top surface, and b) a printedmaterial component, wherein the printed material component compriseswritten instructions for installing the panel onto a top cover (or lidenclosure).

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a perspective view of an exemplary synthesizer.

FIG. 2 illustrates a cross-sectional view of an exemplary synthesizer.

FIG. 3 illustrates a perspective view of a cartridge, chamber bowl andchamber seal of the present invention.

FIG. 4 illustrates a detailed view of an exemplary cartridge.

FIG. 5 illustrates an exemplary drain plate.

FIG. 6A illustrates a top view of one embodiment of a drain plate.

FIG. 6B illustrates a top view of another embodiment of a drain plategasket.

FIG. 7 illustrates a side view of a drain plate gasket situated betweena cartridge and a drain plate.

FIG. 8 illustrates a cross-sectional view of a waste tube system.

FIG. 9 illustrates a chamber bowl with chamber drain.

FIG. 10 illustrates one embodiment of a synthesis column.

FIG. 11 illustrates a computer system coupled to a synthesizer.

FIGS. 12A-C illustrate 3 cross-sectional detailed views of differentembodiments of a cartridge, drain plate, drain plate gasket, receivinghole of cartridge, and synthesis column.

FIGS. 13A and 13B illustrate embodiments of reagent dispense stations.

FIG. 14 illustrates a branched delivery component.

FIG. 15 illustrates an exemplary waste disposal system

FIG. 16 illustrates a synthesizer 1, a robotic means 92, a cleave anddeprotect component 93 and a purification component 94.

FIGS. 17A-C illustrate different embodiments of energy input components95 and mixing components 96.

FIGS. 18A-B illustrate different combinations of energy input components95 and mixing components 96.

FIG. 19A illustrates a synthesizer having a ventilation opening in a lidenclosure

FIGS. 19B and 19C illustrate a synthesizer having ventilation tubingattached to a ventilation opening in a lid enclosure.

FIGS. 20A and 20B illustrate synthesizers having ventilated workspaces.

FIGS. 21A and 21B provide cross sectional views of an exemplarysynthesizer having a lid enclosure 102, and illustrate air flow 109toward the ventilation tubing 103 when the lid enclosure 102 is in aclosed or opened position, respectively.

FIGS. 22A and 22B provide cross sectional views of an exemplarysynthesizer having a primarily enclosed space in a base 2, andillustrate air flow 109 toward the ventilation tubing 103 when the lidenclosure 102 is in a closed or opened position, respectively.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to nucleic acid synthesizers and methodsof using and modifying nucleic acid synthesizers. For example, thepresent invention provides highly efficient, reliable, and safesynthesizers that find use, for example, in high throughput andautomated nucleic acid synthesis (e.g. arrays of synthesizers), as wellas methods of modifying pre-existing synthesizers to improve efficiency,reliability, and safety.

A problem with currently available synthesizers is the emission ofundesirable gaseous or liquid materials that pose health, environmental,and explosive hazards. Such emissions result from both the normaloperation of the instrument and from instrument failures. Emissions thatresult from instrument failures cause a reduction or loss of synthesisefficiency and can provoke further failures and/or complete synthesizerfailure. Correction of failures may require taking the synthesizeroff-line for cleaning and repair. The present invention provides nucleicacid synthesizers with components that reduce or eliminate unwantedemissions and that compensate for and facilitate the removal of unwantedemissions, to the extent that they occur at all. The present inventionalso provides waste handling systems to eliminate or reduce exposure ofemissions to the users or the environment. Such systems find use withindividual synthesizers, as well as in large-scale synthesis facilitiescomprising many synthesizers (e.g. arrays of synthesizers).

In some particularly preferred embodiments, the present inventionprovides efficient and safe “open system synthesizers.” Open systemsynthesizers are contrasted to “closed system synthesizers” in that thereagent delivery, synthesis compartments, and waste extraction for eachsynthesis column are not contained in a system that remains physicallyclosed (i.e., closed from both the ambient environment and from theother synthesis columns in the same instrument) for the duration of thesynthesis run. For example, in a closed system, tubing (or other means)provided for the addition and removal of reagent to each reactioncompartment or synthesis column is generally fixed to the column with acoupling that is sealed to isolate the contents of that system from itssurroundings. In contrast, in an open system, the dispensing and/orremoval of reagent may be through means that are not physically coupledto the reaction compartment.

Further, a common dispensing or waste removal means may be shared bymultiple reaction compartments, such that each compartment sharing themeans is serviced in turn. An example of an “open system synthesizer” isdescribed in PCT Publication WO 99/65602, herein incorporated byreference in its entirety. This publication describes a rotarysynthesizer for parallel synthesis of multiple oligonucleotides. Thetubing that supplies the synthesis reagents to the synthesis column doesnot form a continuous closed seal to the synthesis columns. Instead, therotor turns, exposing the synthesis columns, in series, to the dispenselines, which inject synthesis reagents into the synthesis column. Opensynthesizers offer advantages over closed synthesizers for thesimultaneous production of multiple oligonucleotides. For example, alarge number of independent synthesis columns, each intended to producea distinct oligonucleotide, are exposed to a smaller number of dedicatedreagent dispensers (e.g., four dedicated dispensers for each of thenucleotides). Open systems also provide easy access to synthesiscolumns, which can be added or removed without detaching any otherwisefixed connections to reagent dispensing tubing.

While open synthesizers have advantages for the production ofoligonucleotides, they suffer from increased problems of emissions andfailures. The direct exposure of the columns to their surroundings andthe non-continuous path of reagents increases the number of points atwhich gaseous and liquid emissions occur, thereby increasing the releaseof unwanted emissions to the atmosphere and leakage within thesynthesizer. Many synthesizers carry out reagent delivery, nucleic acidsynthesis, and waste disposal under pressurized conditions. Open systemshave frequent problems with loss of pressure, resulting in instrumentfailures and/or loss of synthesis efficiency. The open systemsynthesizers of the present invention dramatically reduce instrumentfailures and the corresponding emissions.

Whether a system used is open or closed, oligonucleotide synthesisinvolves the use of an array of hazardous materials, including but notlimited to methylene chloride, pyridine, acetic anhydride, 2,6-lutidine,acetonitrile, tetrahydrofurane, and toluene. These reagents can have avariety of harmful effects on those who may be exposed to them. They canbe mildly or extremely irritating or toxic upon short-term exposure;several are more severely toxic and/or carcinogenic with long-termexposure. Many can create a fire or explosion hazard if not properlycontained. In addition, many of these chemicals must be assessed foremissions from normal operations, e.g. for determining compliance withOSHA or environmental agency standards. Malfunction of a system, e.g.,as recited above, increases such emissions, thereby increasing the riskof operator exposure, and increasing the risk that an instrument mayneed to be shut down until risk to an operator is reduced and until anyregulatory requirements for operation are met.

Emission or leakage of reagents during operation can have consequencesbeyond risks to personnel and to the environment. As noted above,instruments may need to be removed from operation for cleaning, leadingto a temporary decrease in production capacity of a synthesis facility.Further, any emission or leakage may cause damage to parts of theinstrument or to other instruments or aspects of the facility,necessitating repair or replacement of any such parts or aspects,increasing the time and cost of bringing an instrument back intooperation. Failure to address emissions or leakage concerns may lead toadditional expenses for operation of a facility, e.g., costs forincreased or improved fire or explosion containment measures, andaddition of costs associated with the elimination of any instrumentsystems or wiring that have not been determined to be safe for use insuch hazardous locations (e.g., by reference to controlling codes, suchas electrical codes, or codes covering operations in the presence offlammable and combustible liquids).

The synthesizers of the present invention provide a number of novelfeatures that dramatically improve synthesizer performance and safetycompared to available synthesizers. These novel features work bothindependently and in conjunction to provide enhanced performance. Forexample, in some embodiments, the synthesizers of the present inventionprevent loss of pressure during synthesis and waste disposal. Bypreventing loss of pressure, synthesis columns are purged properly anddo not overflow during subsequent synthesis steps. Thus, prevention ofpressure loss further prevents liquid overflow and instrumentcontamination. Additionally, in some embodiments, sufficient pressuredifferentials are maintained across all columns to allow efficientsynthesis and purging without instrument failure. For example,regardless of whether synthesis columns are actively involved in aparticular round of synthesis (e.g., short oligonucleotides will becompleted prior to the completion of longer oligonucleotides and willnot be actively synthesized during the later round of synthesis),sufficient pressure differentials are maintained to allow reagentdelivery and purging from the active columns. A number of additionalfeatures of the synthesizers of the present invention are described indetail below.

In addition to providing efficient synthesizers, the present inventionprovides methods for modifying existing synthesizers to improve theirefficiency. For example, one or more of the novel components of thepresent invention may be added into or substituted into existingsynthesizers to improve efficiency and performance.

The present invention further provides means of reducing exposure ofoperators and the environment to synthesis reagents and waste. In oneembodiment, the present invention reduces exposure by improvingcollection and disposal of emissions that occur during the normaloperation of various synthesis instruments. In another embodiment, thepresent invention reduces exposure by improving aspects of theinstrument to reduce risk of malfunctions leading to reagent escape fromthe system, e.g., through leakage, overflow or other spillage. In yetanother embodiment, the present invention reduces exposure by providingan integrated ventilation system, e.g. an integrated fume hood, suchthat even when an instrument is opened (e.g., when the lid or top coveris opened), fumes are collected into a ventilation system, therebyreducing emission into the ambient environment.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “coupled,” as in “coupled attachment,” refersto attachments between objects that do not, by themselves, provide apressure-tight seal. For example, two metal plates that are attached byscrews or pins may comprise a coupled attachment. While the two platesare attached, the seam between them does not form a pressure-tight seal(i.e., gas and/or liquid can escape through the seam).

As used herein, the terms “centralized control system” or “centralizedcontrol network” refer to information and equipment management systems(e.g., a computer processor and computer memory) operable linked to amodule or modules of equipment (e.g., DNA synthesizer or a computeroperably linked to a DNA synthesizer).

As used herein the terms “processor” and “central processing unit” or“CPU” are used interchangeably and refer to a device that is able toread a program from a computer memory (e.g., ROM or other computermemory) and perform a set of steps according to the program.

As used herein, the terms “computer memory” and “computer memory device”refer to any storage media readable by a computer processor. Examples ofcomputer memory include, but are not limited to, RAM, ROM, computerchips, digital video disc (DVDs), compact discs (CDs), hard disk drives(HDD), flash (solid state) recording media and magnetic tape.

As used herein, the term “synthesis and purge component” refers to acomponent of a synthesizer containing a cartridge for holding one ormore synthesis columns attached to or connected to a drain plate forallowing waste or wash material from the synthesis columns to bedirected to a waste disposal system.

As used herein, the term “cartridge” refers to a device for holding oneor more synthesis columns. For example, cartridges can contain aplurality of openings (e.g., receiving holes) into which synthesiscolumns may be placed. “Rotary cartridges” refer to cartridges that, inoperation, can rotate with respect to an axis, such that a synthesiscolumn is moved from one location in a plane (a reagent dispensinglocation) to another location in the plane (a non-reagent dispensinglocation) following rotation of the cartridge.

As used herein, the term “nucleic acid synthesis column” or “synthesiscolumn” refers to a container or chamber in which nucleic acid synthesisreactions are carried out. For example, synthesis columns includeplastic cylindrical columns and pipette tip formats, containing openingsat the top and bottom ends. The containers may contain or provide one ormore matrices, solid supports, and/or synthesis reagents necessary tocarry out chemical synthesis of nucleic acids. For example, in someembodiments of the present invention, synthesis columns contain a solidsupport matrix on which a growing nucleic acid molecule may besynthesized. Nucleic acid synthesis columns may be providedindividually; alternatively, several synthesis columns may be providedtogether as a unit, e.g., in a strip or array, or as device such as aplate having a plurality of suitable chambers. Columns may beconstructed of any material or combination of materials that do notadversely affect (e.g., chemically) the synthesis reaction or the use ofthe synthesized product. For example, columns or chambers may comprisepolymers such as polypropylene, fluoropolymers such as TEFLON, metalsand other materials that are substantially inert to synthesis reactionconditions, such as stainless steel, gold, silicon and glass. In someembodiments, chambers comprise a coating of such a suitable materialover a structure comprising a different material.

As used herein, the term “seal” refers to any means for preventing theflow of gas or liquid through an opening. For example, a seal may beformed between two contacted materials using grease, o-rings, gaskets,and the like. In some embodiments, one or both of the contactedmaterials comprises an integral seal, such as, e.g., a ridge, a lip oranother feature configured to provide a seal between said contactedmaterials. An “airtight seal” or “pressure tight seal” is a seal thatprevents detectable amounts of air from passing through an opening. A“substantially airtight” seal is a seal that prevents all but negligibleamounts of air from passing through an opening. Negligible amounts ofair are amounts that are tolerated by the particular system, such thatdesired system function is not compromised. For example, a seal in anucleic acid synthesizer is considered substantially airtight if itprevents gas leaks in a reaction chamber, such that the gas pressure inthe reaction chamber is sufficient to purge liquid in synthesis columnscontained in the reaction chamber following a synthesis reaction. If gaspressure is depleted by a leak such that synthesis columns are notpurged (e.g., resulting in overflow during subsequent synthesis rounds),then the seal is not a substantially airtight seal. A substantiallyairtight seal can be detected empirically by carrying out synthesis andchecking for failures (e.g., column overflows) during one or a series ofreactions.

As used herein, the term “sealed contact point” refers to sealed seamsbetween two or more objects. Seals on sealed contact points can be ofany type that prevent the flow of gas or liquid through an opening. Forexample, seals can sit on the surface of a seam (e.g., a face seal) orcan be placed within a seam, such that a circumferential contact iscreated within the seam.

As used herein, the term “alignment detector” refers to any means fordetecting the position of an object with respect to another object orwith respect to the detector. For example, alignment detectors maydetect the alignment of a dispensing end of a dispensing device (e.g., areagent tube, a waste tube, etc.) to a receiving device (e.g., asynthesis column, a waste valve, etc.). Alignment detectors may alsodetect the tilt angle of an object (e.g., the angle of a plane of anobject with respect to a reference plane). For example, the tilt angleof a plate mounted on a shaft may be detected to ensure a properperpendicular relationship between the plate and the shaft. Alignmentdetectors include, but are not limited to, motion sensors, infra-red orLED-based detectors, and the like.

As used herein, the term “alignment markers” refers to reference pointson an object that allow the object to be aligned to one or more otherobjects. Alignment markers include pictorial markings (e.g., arrows,dots, etc.) and reflective markings, as well as pins, raised surfaces,holes, magnets, and the like.

As used herein, the term “motor connector” refers to any type ofconnection between a motor and another object. For example a motordesigned to rotate another object may be connected to the object througha metal shaft, such that the rotation of the shaft, rotates the object.The metal shaft would be considered a motor connector.

As used herein, the term “packing material” refers to material placed ina passageway (e.g., a synthesis column) in a manner such that itprovides resistance against a pressure differential between the two endsof the passageway (i.e. hinders the discharge of the pressuredifferential). Packing material may comprise a single material ormultiple materials. For example, in some embodiments of the presentinvention, packing material comprising a nucleic acid synthesis matrix(e.g., a solid support for nucleic acid synthesis such as controlledpore glass, polystyrene, etc.) and/or one or more frits are used insynthesis columns to maintain a pressure differential between the twoends of the synthesis column. Packing material may be distributed intothe reaction chambers in a variety of forms. For example, synthesissupport matrix may be provided as a granular powder. In someembodiments, support matrix may be provided in a “pill” form, wherein anappropriate amount of a support material is held together with a binderto form a pill, and wherein one or more pills are provided to a reactionchamber, as appropriate for the scale of the intended reaction, andfurther wherein the binder is removed or inactivated (e.g., during awash step) to allow the powdered matrix to function in the same manneras an unbound powder. The use of a pill embodiment provides theadvantages of facilitating the process of pre-measuring synthesissupport materials, allowing easy storage of support matrices in apre-measured form, and simplifying provision of measured amounts ofsynthesis support matrix to a reaction chamber.

As used herein, the term “idle,” in reference to a synthesis column,refers to columns that do not take part in a particular synthesisreaction step of a nucleic acid synthesizer. Idle synthesis columnsinclude, but are not limited to, columns in which no synthesis occurs atall, as well as columns in which synthesis has been completed (e.g., forshort oligonucleotide) while other columns are actively undergoingadditional synthesis steps (e.g., for longer oligonucleotides).

As used herein, the term “active,” in reference to a synthesis column,refers to columns that take part (or are taking part) in a particularsynthesis reaction step of a nucleic acid synthesizer. Active synthesiscolumns include, but are not limited to, columns in which liquidreagents are being dispensed into, or columns that contain liquidreagents (e.g. waiting to be purged), or columns that are in the processof being purged.

As used herein, the term “O-ring” refers to a component having acircular or oval opening to accommodate and provide a seal aroundanother component having a circular or oval external cross-section. AnO-ring will generally be composed of material suitable for providing aseal, e.g., a resilient air-or moisture-proof material. In someembodiments, an O-ring may be a circular opening in a larger gasket. Asingle gasket may contain multiple openings and thus provide multipleO-rings. In other embodiments, an O-ring may be ring-shaped, i.e., itmay have circular interior and exterior surfaces that are essentiallyconcentric.

As used herein, the term “viewing window” refers to any transparentcomponent configured to allow visual inspection of an item or materialthrough the window. An enclosure may include a transparent portion thatprovides a viewing window for item within the disclosure. Likewise, anenclosure may be made entirely of a transparent material. In suchembodiments, the entire enclosure can be considered a viewing window. A“viewing window” in an enclosure that is “configured to allow visualinspection” of items in the enclosure “without opening the enclosure”refers to a viewing window in an enclosure of sufficient size, location,and transparency to allow the item to be viewed, unhindered, by thehuman eye. For example, where the item is one or more reagent bottles,the window is configured to allow viewing of the reagents bottles by thehuman eye to determine if the bottles or full or empty. A window thatdoes not provide adequate visual inspection of each of the reagentbottles is not configured to allow visual inspection of reagents in theenclosure without opening the enclosure.

As used herein, the term “enclosure” refers to a container thatseparates materials contained in the enclosure from the ambientenvironment (e.g., as in a sealed system). For example, an enclosure maybe used with a reagent station to contain reagents within an interiorchamber of the enclosure, and therefore separate the reagents from theambient environment. In some embodiments, the enclosure provides anairtight or substantially airtight seal between the interior andexterior of the enclosure. The enclosure may contain one or more valves(e.g., ventilation ports), doors, or other means for allowing gasses orother materials (e.g., reagent bottles) to enter or leave the interiorenvironment of the enclosure.

As used herein, the term “reaction enclosure” refers to an enclosurethat separates the reaction columns or other reaction vessels (e.g.,microplates) from the ambient environment. For example, a chamber bowl18 closed with a top cover 30 and sealed with a chamber seal 31 is oneexemplary embodiment of a reaction enclosure. Another example of areaction enclosure is a synthesis case, e.g., as provided with aPOLYPLEX synthesizer (GeneMachines, San Carlos, Calif.) and with thesynthesizers described in WO 00/56445. In preferred embodiments,reaction enclosures can be sealed during at least one step of operation(e.g., during active synthesis) and can be opened for at least one stepof operation (e.g., for inserting or removing reaction vessels).

As used herein, the term “top enclosure” refers to an enclosure thatforms a primarily enclosed space over the top cover. In preferredembodiments, the top enclosure has four sides (e.g., four top enclosuresides, e.g., 98) and a top panel (e.g., 97) that form a primarilyenclosed space (e.g. 104) above the top cover (e.g., 30) containing aplurality of valves (e.g., 10) and a plurality of dispense lines (e.g.,6). In some embodiments, the primarily enclosed space (e.g., 104) isopen to the ambient environment through a ventilation slot (e.g., 100)in the top cover or the top enclosure. In certain embodiments, the toppanel (e.g., 99) contains an outer window (e.g., 101).

Also as used herein, the combination of a “top enclosure” and “topcover” (e.g., formed as one unit, or connected together) is referred tocollectively as the “lid enclosure”. In preferred embodiments, the “lidenclosure” (e.g., 102) has six sides, with the top cover (e.g., 30)serving as the “bottom”, the top panel serving as the surface oppositethe top cover, and the four side walls being the top enclosure sides(e.g., 98). In certain embodiments, the lid enclosure is hinged so thatis may be moved upward and downward.

As used herein, the term “primarily enclosed space” refers to a spacehaving reduced contact with the ambient environment. A primarilyenclosed space need not be sealed. For example, in some embodiments, aprimarily enclosed space 104 of a lid enclosure of the present inventionhas contact with the ambient environment through a ventilation slot(e.g., 100). In some embodiments, a primarily enclosed space 104 of asynthesizer base 2 has contact with the ambient environment through aventilation slot (e.g., 100)

As used herein, the term “ventilated workspace” refers to a work areathat is open to the ambient environment but that is maintained undernegative air pressure such that air flows into the ventilated workspace,thereby reducing or preventing the flow of fumes and emissions from theventilated workspace into the ambient environment. One example of aventilated workspace is a fume hood (e.g. a chemical fume hood). In someembodiments, the ventilated workspace that is part of an apparatus(e.g., a nucleic acid synthesizer), such that the negative air pressureis maintained over a reaction chamber to draw air away from the reactionchamber so as to prevent the air from entering the ambient environment.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention will be described with reference to severalspecific embodiments, the description is illustrative of the presentinvention and is not to be construed as limiting the invention. Variousmodifications to the present invention can be made without departingfrom the scope and spirit of the present invention. For example, much ofthe following description is provided in the context of an open systemsynthesizer (see, e.g., WO99/65602). However, the invention is notlimited to open system synthesizers.

In preferred embodiments, the present invention provides open-systemsolid phase synthesizers that are suitable for use in large-scalepolymer production facilities. Each synthesizer is itself capable ofproducing large volumes of polymers. However, the present inventionprovides systems for integrating multiple synthesizers into a productionfacility, to further increase production capabilities. The descriptionis provided in the following sections: I) Synthesizers and II)Production Facilities

I) Synthesizers

FIG. 1 illustrates a synthesizer 1. The synthesizer 1 is designed forbuilding a polymer chain by sequentially adding polymer units to a solidsupport in a liquid reagent. The liquid reagents used for synthesizingoligonucleotides may vary, as the successful operation of the presentinvention is not limited to any particular coupling chemistry. Examplesof suitable liquid reagents include, but are not limited to:Acetonitrile (wash); 2.5% dichloroacetic acid in methylene chloride(deblock); 3% tetrazole in acetonitrile (activator); 2.5% cyanoethylphosphoramidite in acetonitrile (A, C, G, T); 2.5% iodine in 9% water,0.5% pyridine, 90.5% THF (oxidizer); 10% acetic anhydride intetrahydrofuran (CAP A); and 10% 1-methylimidazole, 10% pyridine, 80%THF. Various useful reagents and coupling chemistries are described inU.S. Pat. No. 5,472,672 to Bennan, and U.S. Pat. No. 5,368,823 to McGrawet al. (both of which are herein incorporated by reference in theirentireties).

The solid support generally resides within a synthesis column andvarious liquid reagents are sequentially added to the synthesis column.Before an additional liquid reagent is added to a synthesis column, theprevious liquid reagent is preferably purged from the synthesis column.Although the synthesizer 1 is particularly suited for building nucleicacid sequences, the synthesizer 1 is also configured to build any otherdesired polymer chain or organic compound (e.g. peptide sequences).

The synthesizer 1 preferably comprises at least one bank of valves andat least one bank of synthesis columns. Within each bank of synthesiscolumns, there is at least one synthesis column for holding the solidsupport and for containing a liquid reagent such that a polymer chaincan be synthesized. Within the bank of valves, there are preferably aplurality of valves configured for selectively dispensing a liquidreagent into one of the synthesis columns. The synthesizer 1 ispreferably configured to allow each bank of synthesis columns to beselectively purged of the presently held liquid reagent. In particularlypreferred embodiments, the synthesizer of the present invention isconfigured to allow synthesis columns within a bank to be purged evenwhen not all of the synthesis columns contain liquid reagents (e.g. onlya portion of the synthesis columns in a bank received a liquid reagent(i.e. “active”), while the remaining synthesis columns are no longerreceiving liquid reagent (i.e. “idle”). For example, in some preferredembodiments of the present invention, the design of the material in thesynthesis columns allows idle columns to resist the downward pressure ofgas, thus making this pressure available to purge the synthesis columnsthat contain liquid reagent. Additional banks of valves provide thesynthesizer 1 with greater flexibility. For example, each bank of valvescan be configured to distribute liquid reagents to a particular bank ofsynthesis columns in a parallel fashion to minimize the processing time.

Multiple banks of valves can also be configured to distribute liquidreagents to a particular bank of synthesis columns in series. Thisallows the synthesizer 1 to hold a larger number of different reagents,thus being able to create varied nucleic acid sequences (e.g. 48oligonucleotides, each with a unique sequence).

FIG. 1 illustrates a top view of a rotary synthesizer 1. As illustratedin FIG. 1, the synthesizer 1 includes a base 2, a cartridge 3, a firstbank of synthesis columns 4, a second bank of synthesis columns 5, aplurality of dispense lines 6, a plurality of fittings 7 (a first bankof fittings 13, and a second bank of fittings 14), a first bank ofvalves 8 and a second bank of valves 9. Within each of the banks ofvalves 8 and 9, there is preferably at least one valve. Within each ofthe banks of synthesis columns 4 and 5, there is preferably at least onesynthesis column. Each of the valves is capable of selectivelydispensing a liquid reagent into one of the synthesis columns. Each ofthe synthesis columns is preferably configured for retaining a solidsupport such as polystyrene or CPG and holding a liquid reagent.Further, as each liquid reagent is sequentially deposited within thesynthesis column and sequentially purged therefrom, a polymer chain isgenerated (e.g. nucleic acid sequence).

Preferably, there is a plurality of reservoirs, each containing aspecific liquid reagent to be dispensed to one of the plurality ofvalves 8 or 9. Each of the valves within the first bank and second bankof valves 8 and 9, is coupled to a corresponding reservoir. Each of theplurality of reservoirs is pressurized (e.g. by argon gas). As a result,as each valve is opened, a particular liquid reagent from thecorresponding reservoir is dispensed to a corresponding synthesiscolumn. Each of the plurality of dispense lines 6 is coupled to acorresponding one of the valves within the first and second banks ofvalves 8 and 9. Each of the plurality of dispense lines 6 provides aconduit for transferring a liquid reagent from the valve to acorresponding synthesis column. Each one of the plurality of dispenselines 6 is preferably configured to be flexible and semi-resilient innature. In preferred embodiments, the dispense lines of the presentinvention have a large bore size to prevent clogging. In preferredembodiments, the internal diameter of the dispense tube is at least 0.25mm. In other embodiments, the internal diameter of the tube is at least0.50 mm or at least 0.75 mm. In some embodiments, the internal diameterof the tube is greater than or equal to 1.0 mm (e.g. 1.0 mm, or 1.2 mm,or 1.4 mm). Preferably, the plurality of dispense lines 6 are each madeof a material such as PEEK, glass, or coated with TEFLON or Parlene, orcoated/uncoated stainless steel or other metallic material. Of courseother materials may also be used. For example, useful characteristics ofthe material used for the dispense lines would be resistance todegradation by the liquid reagents, minimal “wetting” by the liquidreagents, ease of fabrication, relative rigidity, and ability to beproduced with a smooth surface finish. Metallic tubing (e.g. stainlesssteel), benefit from electropolishing to improve the surface finish(e.g. in coated or uncoated application). Another importantcharacteristic of useful dispense lines in the ability to provide a sealbetween the plurality of valves 10 and the plurality of fittings 7.

Each of the plurality of fittings 7 is preferably coupled to one of theplurality of dispense lines 6. The plurality of fittings 7 arepreferably configured to prevent the reagent from splashing outside thesynthesis column as the reagent is dispensed from the fitting to aparticular synthesis column positioned below the fitting. In preferredembodiments, the fitting includes a nozzle that prevents reagents fromdrying at the point fluid exits the nozzle (e.g. prevents dried reagentsfrom causing the reagents stream to dispense at angles away from theintended synthesis column). Construction techniques to achieveconsistent flow at the discharge point of the liquid reagents isachieved by the use of high quality parts and construction. For example,clean square cuts (without burrs or shavings), or the use of a “drawntip” (i.e., a tip of reduced diameter at the discharge point). The useof a drawn tip, for example, reduces the wall thickness at the point ofdischarge, thus reducing the area of the tube wall cross section,providing a smooth transition from the larger portion of the tube(reducing flow resistance) and increases the likelihood of a cleanseparation of the discharged liquid reagent from the tip of the tube.This clean “snap” of the liquid reagent minimizes the retention of thedischarged fluid at the tip, and thus minimizes subsequent build up ofany solids (e.g. dried reagent). Additionally, if a sharp cut off of thefluid flow is obtained, the fluid front will actually reside within theconfines of the tube after discharge of the desired volume. Thisminimizes surface evaporation and helps to maintain a clean orifice(e.g. prevent reagent from drying at the tip). Another example of auseful technique to prevent liquid reagent from drying at the dischargepoint is providing a sleeve or sheath over the dispense line to a pointnear the tip (dispense point). This sleeve or sheath is particularlyuseful when employed in conjunction with a relatively flexible dispenseline.

As shown in FIG. 1, the first and second banks of valves 8 and 9 eachhave thirteen valves. In FIG. 1, the number of valves in each bank ismerely for exemplary purposes (e.g. other numbers of valves may beemployed, like 14, 15, 16, 17, etc.).

Each of the synthesis columns within the first bank of synthesis columns4 and the second bank of synthesis columns 5 is presently shown restingin one of a plurality of receiving holes 11 within the cartridge 3.Preferably, each of the synthesis columns within the correspondingplurality of receiving holes 11 is positioned in a substantiallyvertical orientation. Each of the synthesis columns is configured toretain a solid support such as polystyrene or CPG and hold liquidreagent(s). In preferred embodiments, polystyrene is employed as thesolid support. Alternatively, any other appropriate solid support can beused to support the polymer chain being synthesized.

During synthesizer operation, each of the valves selectively dispenses aliquid reagent through one of the plurality of dispense lines 6 andfittings 7. The first and second banks of valves 8 and 9 are preferablycoupled to the base 2 of the synthesizer 1. The cartridge 3 whichcontains the plurality of synthesis columns 12 rotates relative to thesynthesizer 1 and relative to the first and second banks of valves 8 and9. By rotating the cartridge 3, a particular synthesis column 12 ispositioned under a specific valve such that the corresponding reagentfrom this specific valve is dispensed into this synthesis column. Inpreferred embodiments, the cartridge 3 has a home position that allowsthe synthesizer to be properly aligned before operation (such that theliquid reagent is properly dispensed into the synthesis columns).Further, the first and second banks of valves 8 and 9 are capable ofsimultaneously and independently dispensing liquid reagents intocorresponding synthesis columns.

A cross sectional view of synthesizer 1 is depicted in FIG. 2. Asdepicted in FIG. 2, the synthesizer 1 includes the base 2, a set ofvalves 15, a motor 16, a gearbox 17, a chamber bowl 18, a drain plate19, a drain 20, a cartridge 3, a bottom chamber seal 21, a motorconnector 22, a waste tube system 23, a controller 24, and a clearwindow 25. The valves 15 are coupled to base 2 of the synthesizer 1 andare preferably positioned above the cartridge 3 around the outside edgeof the base 2. This set of valves 15 preferably contains fifteenindividual valves which each deliver a corresponding liquid reagent in aspecified quantity to a synthesis column held in the cartridge 3positioned below the valves. Each of the valves may dispense the same ordifferent liquid reagents depending on the user-selected configuration.When more than one valve dispenses the same reagent, the set of valves15 is capable of simultaneously dispensing a reagent to multiplesynthesis columns within the cartridge 3. When the valves 15 eachcontain different reagents, each one of the valves 15 is capable ofdispensing a corresponding liquid reagents to any one of the synthesiscolumns within the cartridge 3.

The synthesizer 1 may have multiple sets of valves. The plurality ofvalves within the multiple sets of valves may be configured in a varietyof ways to dispense the liquid reagents to a select one or more of thesynthesis columns. For example, in one configuration, where each set ofvalves is identically configured, the synthesizer 1 is capable ofsimultaneously dispensing the same reagent in parallel from multiplesets of valves to corresponding banks of synthesis columns. In thisconfiguration, the multiple banks of synthesis columns may be processedin parallel. In the alternative, each individual valve within multiplesets of valves may contain entirely different liquid reagents such thatthere is no duplication of reagents among any individual valves in themultiple sets of valves. This configuration allows the synthesizer 1 tobuild polymer chains requiring a large variety of reagents withoutchanging the reagents associated with each valve.

The motor 16 is preferably mounted to the base 2 through the gear box 17and the motor connector 22. The chamber bowl 18 preferably surrounds themotor connector 22 and remains stationary relative to the base 2.

The chamber bowl 18 is designed to hold any reagent spilled from theplurality of synthesis columns 12 during the purging process (or thedispensing process). Further, the chamber bowl 18 is configured with atall shoulder to insure that spills are contained within the bowl 18.The bottom chamber seal 21 preferably provides a seal around the motorconnector 22 in order to prevent the contents of the chamber bowl 18from flowing into the gear box 17 (see FIG. 9). The bottom chamber seal21 is preferably composed of a flexible and resilient material such asTEFLON (or elastomer which conforms to any irregularities of the motorconnector 22). Alternatively, the bottom chamber seal can be composed ofany other appropriate material. In particularly preferred embodiments,the bottom chamber seal is composed of material that resists constantcontact with liquid reagents (e.g., TEFLON or Parlene). Additionally,the bottom chamber seal 21 may have frictionless properties that allowthe motor connector 22 to rotate freely within the seal. For example,coating this flexible material with TEFLON helps to achieve a lowcoefficient of friction.

The clear window 25 is attached to (formed in) a top cover 30 of thesynthesizer 1 and covers the area above the cartridge 3. The top cover30 of synthesizer 1 seals the top part of the chamber (when in place),and opens up allowing an operator or maintenance person access to theinterior of the synthesizer 1. The clear window 25 in top cover 30allows the operator to observe the synthesizer 1 in operation whileproviding a pressure sealed environment within the interior of thesynthesizer 1. As shown in FIG. 2, there are a plurality of throughholes 26 in the clear window 25 to allow the plurality of dispense lines6 to extend through the clear plate 25 to dispense material into thesynthesis columns located in cartridge 3.

The clear window 25 also includes a gas fitting 27 attachedtherethrough. The gas fitting 27 is coupled to a gas line 28. The gasline 28 preferably continuously emits a stream of inert gas (e.g. Argon)which flows into the synthesizer 1 through the gas fitting 27 andflushes out traces of air and water from the plurality of synthesiscolumns 12 within the synthesizer 1. Providing the inert gas flowthrough the gas fitting 27 into the synthesizer 1 prevents the polymerchains being formed within the synthesis columns from being contaminatedwithout requiring the plurality of synthesis columns 12 to behermetically sealed and isolated from the outside environment.

FIG. 3 shows the cartridge 3 in chamber bowl 18, with the top plate 30removed, thus revealing the top chamber seal 31. Top chamber seal 31 isdesigned to provide a tight seal between top plate 30 and chamber bowl18, such that inert gas applied through clear window 25 does not leak.If the top chamber seal 31 does not function properly, the inert gasleaks out (lowering the pressure in the chamber), thus causing the purgeoperation (that relies on the pressure on the inert gas) to fail. Whenthe purge operation fails, un-purged columns quickly fill up andoverflow. In some embodiments, a V-seal type top chamber seal isemployed to prevent leakage of gas. In some embodiments, the hinges andlatches on top plate 30 (not shown) are precisely machined to providebalanced forces on the top plate 30, such that the top plate 30 fitstightly over the chamber bowl.

FIG. 4 illustrates a detailed view of a cartridge 3 for synthesizer 1.Preferably, the cartridge 3 is circular in shape such that it is capableof rotating in a circular path relative to the base 2 and the first andsecond banks of valves 8 and 9. The cartridge 3 has a plurality ofreceiving holes 11 on its upper surface around the peripheral edge ofthe cartridge 3. Each of the plurality of receiving holes 11 isconfigured to hold one of the synthesis columns 12. The plurality ofreceiving holes 11, as shown on the cartridge 3, is divided up amongfour banks. A bank 32 illustrates one of the four banks on the cartridge3 and contains twelve receiving holes, wherein each receiving hole isconfigured to hold a synthesis column. An exemplary synthesis column 12is shown being inserted into one of the plurality of receiving holes 11.The total number of receiving holes shown on the cartridge 3 includesforty-eight (48) receiving holes, divided into four banks of twelvereceiving holes each. The number of receiving holes and theconfiguration of the banks of receiving holes is shown on the cartridge3 for exemplary purposes only. Any appropriate number of receiving holesand banks of receiving holes can be included in the cartridge 3.Preferably, the receiving holes 11 within the cartridge each have aprecise diameter for accepting the synthesis columns 12, which also eachhave a corresponding precise exterior surface 61 (see FIG. 10) toprovide a pressure-tight seal when the synthesis columns 12 are insertedinto the receiving holes 11. In preferred embodiments, the synthesiscolumn includes a column seal 65 (see FIG. 10), such as a ring seal or aball seal (e.g., a flexible TEFLON ring that flexes on engagement of thesynthesis column in the receiving hole 11). In other preferredembodiments, a seal, such as a ring seal, is provided above or in thereceiving holes 11 (see, e.g., FIG. 12).

FIG. 5 depicts an exemplary drain plate 19 of the synthesizer 1. Thedrain plate 19 is coupled to the motor connector 22 (not shown) throughsecuring holes 33. More specifically, the drain plate 19 is attached tothe motor connector 22, which rotates the drain plate 19 while the motor16 is operating and the gear box 17 is turning. The cartridge 3 and thedrain plate 19 are preferably configured to rotate as a single unit. Thedrain plate 19 is configured to catch and direct the liquid reagents asthe liquid reagents are expelled from the plurality of synthesis columns(during the purging process). During operation, the motor 16 isconfigured to rotate both the cartridge 3 and the drain plate 19 throughthe gear box 17 and the motor connector 22. The bottom chamber seal 21allows the motor connector 22 to rotate the cartridge 3 and the drainplate 19 through a portion of the chamber bowl 18 while still containingspilled reagents in the chamber bowl 18. The controller 24 is coupled tothe motor 16 to activate and deactivate the motor 16 in order to rotatethe cartridge 3 and the drain plate 19. The controller 24 (see FIGS. 2and 11) provides embedded control to the synthesizer and controls notonly the operation of the motor 16, but also the operation of the valves15 and the waste tube system 23.

The drain plate 19 has a plurality of securing holes 33 for attaching tothe motor connector 22. The drain plate 19 also has a top surface 34which may, in some embodiments, attach to the underside of the cartridge3. In other embodiments, a drain plate gasket is provided between thedrain plate 19 and cartridge 3 (see below). As stated previously, thecartridge 3 holds the plurality of synthesis columns grouped into aplurality of banks. The drain plate preferably has a collection areacorresponding to each of the banks of synthesis columns (e.g. four inFIG. 5 to correspond to the four banks of synthesis columns in cartridge3). Each of these four collection areas 35, 36, 37 and 38 in FIG. 5,forms a recessed area below the top surface 34 and is designed tocontain and direct material flushed from the synthesis columns withinthe bank above the collection area.

Each of the four collection areas 35, 36, 37 and 38 is positioned belowa corresponding one of the banks of synthesis columns on the cartridge3. The drain plate 19 is rotated with the cartridge 3 to keep thecorresponding collection area below the corresponding bank.

In FIG. 5, there are four drains 39, 40, 41, and 42 each of which islocated within one of the four collection areas 35, 36, 37 and 38respectively. In use, the collection areas are configured to containmaterial flushed from corresponding synthesis columns and pass thatmaterial through the drains. Preferably, there is a collection area anda drain corresponding to each bank of synthesis columns within thecartridge 3. Alternatively, any appropriate number of collection areasand drains can be included within a drain plate. FIG. 6A shows a topview of drain plate gaskets 43. The drain plate gasket is configured tobe situated between drain plate 19 and cartridge 3. Drain plate gasket43 is shown in FIG. 6A with guide holes 44 and drain cut-outs 57, 58,59, and 60. Guide holes 44 allow the drain plate gasket to fit over themotor connector 22. Drain cut-outs 57-60 allow the bottom column openingof synthesis columns 12 to discharge material into collection areas35-38 in drain plate 19. In other embodiments, the drain cut outs mirrorthe receiving holes in the cartridge (see cut-outs 60 in FIG. 6B), suchthat each column is able to discharge material into collection areas35-38, while having a seal around each synthesis column. In someembodiments, all of the cut-outs are for the synthesis columns, like thecuts 60 depicted in FIG. 6B.

The drain plate gaskets of the present invention may be made of anysuitable material (e.g. that will provide a tight seal above drain plate19, such that gas and liquid do not escape). In some embodiments, thedrain plate gasket is composed of rubber. Providing a tight seal betweencartridge 3 and drain plate 19 with a drain plate gasket helps maintainthe proper pressure of inert gas during purging procedures, such thatsynthesis columns with liquid reagent properly drain (preventingoverflow during the next cycle). The seal between cartridge 3 and drainplate 19 may also be improved by the addition of grease between thecomponents, or very finely machining the contact points between the twocomponents. In other embodiments, the seal between the cartridge anddrain plate is improved by physically bonding the plates together, ormachining either the cartridge or drain plate such that concentric ringseals may inserted into the machined component. In still otherembodiments, the two components are manufactured as a single component(e.g. a single components with all the features of both the cartridgeand drain plate formed therein). In preferred embodiments, one componentis provided with plurality of concentric circular rings that contact theflat surface of the other component and act as seals.

FIG. 7 shows a side view of a drain plate gasket 43 situated betweencartridge 3 and drain plate 19. FIG. 7 also shows a drain 20 extendingfrom drain plate 19. FIG. 7 also shows a drain with sealing ring 45(sealing ring is labeled 46). The sealing ring 46 tightly seals theconnection between the drain 45 and the waste tube system 23 (see FIG.8). Also shown in FIG. 7 is a synthesis column 12 inserted in cartridge3, passing through drain plate gasket 43, and ending in drain plate 19.

The waste tube system 23 is preferably utilized to provide a pressurizedenvironment for flushing material including reagents from the pluralityof synthesis columns located within a corresponding bank of synthesiscolumns and expelling this material from the synthesizer 1.Alternatively, the waste tube system 23 can be used to provide a vacuumfor drawing material from the plurality of synthesis columns locatedwithin a corresponding bank of synthesis columns.

A cross-sectional view of the waste tube system 23 is illustrated inFIG. 8. The waste tube system 23 comprises a stationary tube 47 and amobile waste tube 48. The stationary tube 47 and the mobile waste tube48 are slidably coupled together. The stationary tube 47 is attached tothe chamber bowl 18 and does not move relative to the chamber bowl (seeFIG. 9). In contrast, the mobile tube 48 is capable of sliding relativeto the stationary tube 47 and the chamber bowl 18. When in an inactivestate, the waste tube system 47 does not expel any reagents. During theinactive state, both the stationary tube 47 and the mobile tube 48 arepreferably mounted flush with the bottom portion of the chamber bowl 18(see FIG. 9). When in an active state, the waste tube system 23 purgesthe material from the corresponding bank of synthesis columns. Duringthe active state, the mobile tube 48 rises above the bottom portion ofthe chamber bowl 18 towards the drain plate 19. The drain plate 19 isrotated over to position a drain corresponding to the bank to beflushed, above the waste tube system 23. The mobile tube 48 then couplesto the drain (e.g., 20 or 45) and the material is flushed out of thecorresponding bank of synthesis columns and into the drain plate 19. Theliquid reagent is purged from the corresponding bank of synthesiscolumns due to a sufficient pressure differential between a top opening49 (FIG. 10) and a bottom opening 50 (FIG. 10) of each synthesis column.This sufficient pressure differential is preferably created by couplingthe mobile waste tube 48 to the corresponding drain. Alternatively, thewaste tube system 23 may also include a vacuum device 29 (see, FIG. 2)coupled to the stationary tube 47 wherein the vacuum device 29 isconfigured to provide this sufficient pressure differential to expelmaterial from the corresponding bank of synthesis columns. When thissufficient pressure differential is generated, the excess materialwithin the synthesis columns being flushed, then flows through thecorresponding drain and is carried away via the waste tube system 23.

When engaging the corresponding drain to flush a bank of synthesiscolumns, preferably the mobile tube 48 slides over the correspondingdrain such that the mobile tube 48 and the drain act as a single unit.Alternatively, the waste tube system 23 includes a mobile tube 48 whichengages the corresponding drain by positioning itself directly below thedrain and then sealing against the drain without sliding over the drain.The mobile tube 48 may include a drain seal positioned on top of themobile tube. In this embodiment, during a flushing operation, the mobiletube 48 is not locked to the corresponding drain. In the event that thisdrain is accidentally rotated while the mobile waste tube 48 is engagedwith the drain, the drain and mobile tube 48 of the synthesizer 1 willsimply disengage and will not be damaged. If this occurs while materialis being flushed from a bank of synthesis columns, any spillage from thedrain is contained within the chamber bowl 18. In preferred embodiments,the bottom of the chamber bowl 18 has a chamber drain 64 (see FIG. 9) tocollect and remove any spilled material in the chamber bowl. In thisregard, material may be removed before it builds up and leaks into otherparts of the synthesizer (e.g. motor 16 or gear box 17). In someembodiments of the present invention, the chamber drain is in a closedposition during synthesis and purging. When the top cover of thesynthesizer is opened, the chamber drain can be opened, drawing outunwanted gaseous or liquid emissions (e.g., using a vacuum source).Coordination of the chamber drain opening to the top cover opening maybe accomplished by mechanical or electric means.

Configuring the waste tube system 23 to expel the reagent while themobile waste tube 48 is coupled to the drain allows the presentinvention to selectively purge individual banks of synthesis columns.Instead of simultaneously purging all the synthesis columns within thesynthesizer 1, the present invention selectively purges individual banksof synthesis columns such that only the synthesis columns within aselected bank or banks are purged. In preferred embodiments, the wastesystem is fitted for qualitative monitoring of detritylation. Forexample, calorimetric analysis of waste effluent using, for example, aCCD camera or a similar device provides a yes/no answer on a particulardetritylation level. Qualitative analysis can also be accomplished byspectrophotometricly, or by testing effluent conductivity. Qualitativedetection of detritylation can generally be performed with lessexpensive equipment than is generally required by more precisequantitation, and yet generally provides sufficient monitoring fordetritylation failure. In preferred embodiments, the effluent from eachcolumn is monitored when a bank of columns is purged.

Preferably, the synthesizer 1 includes two waste tube systems 23 forflushing two banks of synthesis columns simultaneously. Alternatively,any appropriate number of waste tube systems can be included within thesynthesizer 1 for selectively flushing synthesis columns or banks ofsynthesis columns. In preferred embodiments, the waste tube systems 23are spaced on opposite sides of the chamber bowl 18 (i.e. they aredirectly across from each other, see FIG. 9). In this regard, the forceon the drain plate 19 is equalized during flushing procedures (e.g. thedrain plate is less likely to tip one way or the other from force beingapplied to just one side of the plate). Alternatively, a single wastetube system 23 may be provided for flushing the plurality of banks ofsynthesis columns. When a single waste tube system is used, it ispreferred that a balancing force be provided on the opposite side of thedrain plate 19, e.g., such as would be provided by the presence of asecond waste tube system 23. In one embodiment, a balancing force isprovided by a dummy waste tube system (not shown), that may be actuatedin the same fashion as the waste tube system 23, but which does notserve to drain the bank of synthesis columns to which it is deployed.

In use, the controller 24, which is coupled to the motor 16, the valves15, and the waste tube system 23, coordinates the operation of thesynthesizer 1. The controller 24 controls the motor 16 such that thecartridge is rotated to align the correct synthesis columns with thedispense lines 6 corresponding to the appropriate valves 15 duringdispensing operations and that the correct one of the drains 39, 40, 41,and 42 are aligned with an appropriate waste tube system 23 during aflushing operation.

In some preferred embodiments, the synthesizer comprises a means ofdelivering energy to the synthesis columns to, for example, increasenucleic acid coupling reaction speed and efficiency, allowing increasedproduction capacity. In some embodiments, the delivery of energycomprises delivering heat to the chamber or the columns. In addition toincreasing production capacity, the use of heat allows the use ofalternate synthesis chemistries and methods, e.g., the phosphatetriester method, which has the advantages of using more stable monomerreagents for synthesis, and of not using tetrazole or its derivatives ascondensation catalysts. Heat may be provided by a number of means,including, but not limited to, resistance heaters, visible or infraredlight, microwaves, Peltier devices, transfer from fluids or gasses(e.g., via channels or a jacketed system). In some embodiments, heatgenerated by another component of a synthesis or production facilitysystem (e.g., during a waste neutralization step) is used to provideheat to the chamber or the columns. In other embodiments, heat isdelivered through the use of one or more heated reagents. Delivery ofheat also comprises embodiments wherein heat is created within the,e.g., by magnetic induction or microwave treatment. In some embodiments,heat is created at or within synthesis columns. It is contemplated thatheating may be accomplished through a combination of two or moredifferent means.

In some embodiments, the delivery of heat provides substantially uniformheating to two or more synthesis columns. In some embodiments, heatingis carried out at a temperature in a range of about 20° C. to about 60°C. The present invention also provides methods for determining anoptimum temperature for a particular coupling chemistry. For example,multiple synthesizers are run side-by-side with each machine run at adifferent temperature. Coupling efficiencies are measured and theoptimum temperature for one or more incubations times are determined. Inother embodiments, different amounts of heat are delivered to differentsynthesis columns within a single synthesizer, such that differentreaction chemistries or protocols can be run at the same time.

Delivery of heat to an enclosed, sealed system will alter the pressurewithin the system. It is contemplated that the sealed system of thepresent invention will be configured to tolerate variations in thesystem pressure (i.e., the pressure within the sealed system) related toheating or other energy input to the system. In preferred embodiments,the system (e.g., every component of the system and every junction orseal within the system) will be configured to withstand a range ofpressures, e.g., pressures ranging from 0 to at least 1 atm, or about 15psi. It is contemplated that pressures may be varied between differentpoints within the system. For example, in some embodiments, reagents andwaste fluids are moved through the synthesis column by use of a pressuredifferential between one end (e.g., an input aperture) and the other(e.g., a drain aperture) of the synthesis column. In some embodiments,the system of the present invention is configured to use pressuredifferentials within a pressurized system (e.g., wherein a systemsegment having lower pressure than another system segment nonethelesshas higher pressure than the environment outside the sealed system). Insome embodiments, the prevention of backward flow of reagents throughthe system (e.g., in the event of back pressure from a process step suchas heating) is controlled by use of pressure. In other embodiments,valves are provided to assist in control of the direction of flow.

In other preferred embodiments, the synthesizer comprises a mixingcomponent configured to mix reaction components, e.g., to facilitate thepenetration of reagents into the pores of the solid support. Mixing maybe accomplished by a number of means. In some embodiments, mixing isaccomplished by forced movement of the fluid through the matrix (e.g.,moving it back and forth or circulating it through the matrix usingpressure and/or vacuum, or with a fluid oscillator). Mixing may also beaccomplished by agitating the contents of the synthesis column (e.g.,stirring, shaking, continuous or pulsed ultra or subsonic waves). Insome preferred embodiments, an agitator is used that avoids the creationof standing waves in the reaction mixture. In some preferredembodiments, the agitator is configured to utilize a reaction vesselsurface or reaction support surface (e.g., a surface of a synthesiscolumn) to serve as resonant members to transfer energy into fluidwithin a reaction mixture. In a preferred embodiment, a horn is applieddirectly to the cartridge 3 to provided pulsed or continuous ultra sonicenergy to the synthesis columns therein. In some embodiments, the matrixis an active component of the mixing system. For example, in someembodiments, the matrix comprises paramagnetic particles that may bemoved through the use of magnets to facilitate mixing. In someembodiments, the matrix is an active component of both mixing andheating systems (e.g., paramagnetic particles may be agitated bymagnetic control and heated by magnetic induction). It is contemplatedthat any of these mixing means may be used as the sole means of mixing,or that these mixing components may be used in combination, eithersimultaneously or in sequence. In preferred embodiments, the heatingcomponent and the mixing component are under automated control.

FIG. 10 illustrates a cross sectional view of a synthesis column 12. Thesynthesis column is an integral portion of the synthesizer 1. Generally,the polymer chain is formed within the synthesis column 12. Morespecifically, the synthesis column 12 holds a solid support 54 on whichthe polymer chain is grown. Examples of suitable solid supports include,but are not limited to, polystyrene, controlled pore glass, and silicaglass. As stated previously, to create the polymer chain, the solidsupport 54 is sequentially submerged in various reagents for apredetermined amount of time. With each deposit of a reagent, anadditional unit is added, or the solid support is washed, or failuresequences are capped, etc. Preferably, the solid support 54 is heldwithin the synthesis column 12 by a bottom frit 55. In particularlypreferred embodiments, a top frit 53 is included above the solid support(e.g. to help resist downward gas pressure when the particular synthesiscolumn does not have liquid reagents, but other synthesis columns withinthe bank are being purged of their liquid contents). The synthesiscolumn 12 includes a top opening 49 and a bottom opening 50. During thedispensing process, the synthesis column 12 is filled with a reagentthrough the top opening 49. During the purging process, the synthesiscolumn 12 is drained of the reagent through the bottom opening 50. Thebottom frit 55 prevents the solid support from being flushed away duringthe purging process.

The exterior surface 61 of each synthesis column 12 fits within thereceiving hole 11 within the cartridge 3 and provides a pressure tightseal around each synthesis column within the cartridge 3. Preferably,each synthesis column is formed of polyethylene or other suitablematerial. In preferred embodiments, the receiving holes 11 of thecartridge 3 are provided with seals, such as O-ring seals 67, that willflex on engagement of the synthesis column 12 in receiving hole 11 andaccommodate any irregularities in the exterior surface 61 of thesynthesis column 12, thus assuring the presence of a pressure-tightseal.

In preferred embodiments, the material inside the synthesis column (e.g.in FIG. 10, this includes top frit 53, solid support 54, and bottom frit55) is configured to resist the downward pressure of gas (e.g., toprovide back pressure) applied during the purging process when theparticular synthesis column does not have liquid reagent. In thisregard, other synthesis columns that do contain liquid reagents may besuccessfully purged with the application of gas pressure during thepurging process (i.e. the synthesis columns without liquid reagent donot allow a substantial portion the gas pressure applied during thepurging process to escape through their bottom openings). Other packingmaterials may also be added to the synthesis columns to help maintainthe pressure differential across the column when it is idle.

One method for constructing a synthesis column that successfully resiststhe downward pressure of gas (when no liquid reagent has been added tothis column) is to include a top frit in addition to a bottom frit.Determining what type of top frit is suitable for any given synthesiscolumn and type of solid support may be determined by test runs in thesynthesizer. For example, the columns may be loaded into the synthesizerwith the candidate top frit (and solid support and bottom frit), andinstructions for synthesizing different length oligonucleotides inputted(i.e., this will allow certain columns to sit idle while other columnsare still having liquid dispensed into them and purged out). Observationthrough the glass panel, examining the amount of leakage fromoverflowing columns, and testing the quality of the resultingoligonucleotides, are all methods to determine if the top frit issuitable (e.g., a thicker or smaller pore top frit may be employed ifproblems associated with insufficient back pressure are seen). Bycombining the appropriate packing material in columns with theappropriate delivered pressure to the chamber, purging can beefficiently carried out, avoiding spill-over that can result insynthesis or instrument failure.

Another method for constructing a synthesis column that successfullyresists the downward pressure of gas (when no liquid reagent has beenadded to this column) is to provide a solid support that resists thisdownward force even when no liquid reagent is in the columns. Onesuitable solid support material is polystyrene (e.g. U.S. Pat. No.5,935,527 to Andrus et al., hereby incorporated by reference). In someembodiments, the styrene (of the polystyrene) is cross-linked with across-linking material (e.g. divinylbenzene). In some embodiments, thecross-linking ratio is 10-60 percent. In preferred embodiments, thecross-linking ration is 20-50 percent. In particularly preferredembodiments, the cross-linking ratio is about 30-50 percent. In someembodiments, the polystyrene solid support is used in conjunction with atop frit in order to successfully resist the downward pressure of gasduring the purging process. In some embodiments, the polystyrene is usedas the solid support for synthesis. In other embodiments, a differentsupport, such as controlled pore glass, is used as the support for thesynthesis reaction, and the polystyrene is provided only to increase theback pressure from a column comprising a CPG or other synthesis support.

There are many advantages of configuring synthesis columns tosuccessfully resist downward gas pressure during the purging process.One advantage is the fact that not all the synthesis columns need tocontain liquid reagent during the purging process in order for the purgeto be successful. Instead, one or more of the synthesis columns mayremain idle during a particular cycle, while the other synthesis columnscontinue to receive liquid reagents. In this regard, oligonucleotides ofdifferent lengths may be constructed (e.g., a 20-mer constructed in onesynthesis column may be completed and sit idle, while a 32-mer isconstructed in a second synthesis column). Achieving successful purgesafter each liquid addition prevents liquid leakage (e.g. additionalliquid reagent applied to a synthesis column that was not successfullypurged will cause the column to overflow).

FIG. 11 illustrates a computer system 62 coupled to the synthesizer 11.The computer system 62 preferably provides the synthesizer 1, andspecifically the controller 24, with operating instructions. Theseoperating instructions may include, for example, rotating the cartridge3 to a predetermined position, dispensing one of a plurality of reagentsinto selected synthesis columns through the valves 15 and dispense lines6, flushing the first bank of synthesis columns 4 and/or the second bankof synthesis columns 5, and coordinating a timing sequence of thesesynthesizer functions. U.S. Pat. No. 5,865,224 to Ally et al. (hereinincorporated by reference in its entirety), further demonstratescomputer control of synthesis machines. Preferably, the computer system62 allows a user to input data representing oligonucleotide sequences toform a polymer chain via a graphical user interface.

After a user inputs this data, the computer system 62 instructs thesynthesizer 1 to perform appropriate functions without any further inputfrom the user. The computer system 62 preferably includes a processor,an input device and a display. The computer 62 can be configured as alaptop or a desktop, and may be operably connected to a network (e.g.LAN, internet, etc.).

In some embodiments, the present invention provides alignment detectorsfor detecting the alignment of any of the components of the presentinvention, as desired. In some embodiments, when a misalignment isdetected, an alarm or other signal is provided so that a user can assureproper alignment prior to further operation. In other embodiments, whena misalignment is detected, a processor operates a motor to adjust thatalignment. Alignment detectors find particular use in the presentinvention for assuring the alignment of any components that are involvedin an exchange of liquid materials. For example, alignment of dispenselines and synthesis columns and alignment of drains and waste tubesshould be monitored. Likewise, the tilt angle of the cartridge or anyother component that should be parallel to the work surface can bemonitored with alignment detectors.

As noted above, the exterior surface 61 of each synthesis column 12 fitswithin the receiving hole 11 within the cartridge 3 and is intended toprovide a pressure-tight seal around each synthesis column 12 within thecartridge 3. FIG. 12 illustrates three cross-sectional detailed views ofthe assembly 66 (the assembly comprising the cartridge 3, the drainplate gasket 43 and the drain plate 19) with a synthesis column 12within a receiving hole 11 of cartridge 3. Each view shows a differentembodiment of an airtight seal between the assembly 66 and the exteriorsurface 61 of synthesis column 12. In some embodiments, the airtightseal is provided by an O-ring 67. In preferred embodiments, the O-ring67 is accessible for easy insertion and removal, e.g., for cleaning orreplacement. In one embodiment, an O-ring 67 is positioned at the top ofreceiving hole 11, held in place by, e.g., a restraining plate 68, orany other suitable restraining fitting. In a preferred embodiment, achannel 69 is provided at the top of receiving hole 11 in cartridge 3 toaccommodate the O-ring 67, as illustrated in FIG. 12A. In a particularlypreferred embodiment, a groove 70 within receiving hole 11 in cartridge3 accommodates an O-ring 67, providing a groove lip 71 to restrain theO-ring 67, as illustrated in FIG. 12B. In a particularly preferredembodiment, the groove lip 71 is about 0.030 inches. FIG. 12Cillustrates a further embodiment, in which drain plate gasket 43 isconfigured to provide an airtight seal between nucleic acid synthesiscolumn 12 and assembly 66. The illustrations in FIG. 12 are provided byway of examples only, and it is not intended that the present inventionbe limited by details of these illustrations, such as apparent size,shape or precise locations of features such as grooves, channels, platesor seals. Any O-ring configuration that helps maintain proper pressuredifferential across the synthesis columns is contemplated.

O-rings 67 may be composed of any suitable material, preferably achemically resistant, resilient material that flexes upon engagement ofthe synthesis column 12 in receiving hole 11. In some embodiments, a lowcost material such as silicone or VITON may be used. In otherembodiments, more expensive materials offering longer term stability,such as KALREZ, may be used. In some embodiments the O-rings may have alight lubrication, e.g. with a silicone or fluorinated grease.

In some embodiments, the present invention provides a means ofcollecting emissions from reagent reservoirs 72 (See e.g., FIGS. 13A andB) by providing a reagent dispensing station. In one embodiment, thereagent dispensing station is an integral part of the base 2 of thesynthesizer, as illustrated in FIGS. 13A and 13B. In some embodiments,the reagent dispensing station provides an enclosure for collectingemitted gasses. In some embodiments, the enclosure is created by theprovision of a panel 73 to enclose a portion of base 2 containingreagent reservoirs 72, as illustrated in FIG. 13B. In some embodiments,the panel 73 is movable for easy access to reagent reservoirs. In someembodiments, it is removeably attached. Removable attachment may beaccomplished by any suitable means, such as through the use of VELCRO,screws, bolts, pins, magnets, temporary adhesives, and the like. Inpreferred embodiments, at least a portion of the panel 73 is slidablymoveable. In preferred embodiments, at least a portion of panel 73 istransparent. In some embodiments, the enclosure of the reagentdispensing station comprises a viewing window that is not in a panel 73.

In some embodiments, the enclosure comprises a ventilation tube. Inpreferred embodiments, panel 73 comprises a ventilation port 74, e.g.,for attachment to a ventilation tube. Since reagent vapors are typicallyheavier than air, in preferred embodiments, the ventilation tube isattached at the bottom for the enclosure. In a particularly preferredembodiment, the ventilation port is positioned toward the rear of theinstrument.

In some embodiments, the enclosure further comprises an air inlet. In apreferred embodiment, a clearance 75 between the panel 73 and the base 2provides an air inlet. In a particularly preferred embodiment, the airinlet is positioned toward the front of the instrument.

The location of the ventilation port 74 and air inlet is not limited tothe panel 73. For example, in an alternative embodiment, the reagentdispensing station comprises a stand for holding the reagent bottles anda ventilation tube, wherein the stand holds the reagent reservoirs andthe ventilation tube removes emitted gases.

Ventilation may be continuous or under the control of an operator. Forexample, in some embodiments, when the panel 73 is in a closed position,ventilation occurs continuously through the ventilation port 74 or atregular intervals. In other embodiments, an operator may manuallyactivate ventilation prior to opening the panel 73. In still otherembodiments, ventilation occurs in an automated fashion immediatelyprior to the opening of panel 73. For example, where the opening ofpanel 73 is controlled by a computer processor, activation of the “open”routine triggers ventilation prior to the physical opening of panel 73.In still other embodiments, the contents of the reagent containers aremonitored by a sensor and the ventilation is triggered when one or moreof the reagent containers are depleted. In some embodiments, the panel73 is also automatically open, indicating the need for additionalreagents and/or allowing an automated reagent container delivery systemto supply reagents to the system.

The present invention also provides systems for ventilation,particularly ventilation of reaction enclosures (e.g., a chamber bowl18), that improve the safety of synthesizers. The ventilation systems ofthe present invention may be applied to any type of synthesizer, andpreferably, to open type synthesizers. These systems are particularlyuseful for improving the function and safety of certain commerciallyavailable synthesizers, such as the ABI 3900 Synthesizer.

During normal operations and without any malfunction, fumes arenonetheless are emitted from the chamber bowl of the 3900 machine whenthe synthesizer is opened for access by an instrument operator (e.g.,when the top cover or lid enclosure is opened to retrieve columns aftersynthesis is completed). These emissions can be significant. In someinstances, instruments such as the 3900 may be installed inside chemicalfume hoods to collect such emissionsfrom normal operations. However,placing machines in chemical fume hoods is not practical for a number ofreasons. For example, the presence of a large instrument within achemical fume hood limits the use of the hood for other purposes.Removal of the instrument when the hood is needed for another purpose isimpractical, since many synthesizers are physically connected toexternal reagent reservoirs, gas tanks or other supply sources, makingfrequent removal and reinstallation prohibitively complex. Anotherproblem with using chemical fume hoods to contain and remove emissionsis that, using this approach, the number of synthesizers that can beused at one time is limited by the amount of hood space available. Thisprevents the use of many synthesizers in parallel, e.g., in an array ofsynthesizers, and therefore limits high-throughput synthesis capability.What is needed are systems to properly vent synthesizers, such as the3900, that do not require placing the machines in chemical fume hoods.

The present invention provides systems for collecting emissions fromsynthesizers without the use of a separate fume hood. The presentinvention comprises a synthesizer having an integrated ventilationsystem to contain and remove vapor emissions. By way of example, theintegrated ventilation system of the present invention is described asapplied to the components and features of open synthesizers like theApplied Biosystems 3900 instrument. However, this configuration is usedonly as an example, and the integrated ventilation systems are notintended to be limited to the 3900 instrument or to any particularsynthesizer. One aspect of the invention is to collect and remove vaporswhen the instrument is open, e.g., for access by the operator to thereaction chamber (FIGS., 19C, and 20A-C). In one embodiment of thepresent invention, the integrated ventilation system comprises aventilated workspace. Embodiments of an integrated ventilation systemcomprising a ventilated workspace as applied to the 3900 instrument areshown in FIGS. 19A-C, 20A-C and 21 A-B. Another embodiment is diagrammedin FIGS. 22 A and B.

In some embodiments, a ventilation opening is provided through anopening in the top. For example, referring to FIG. 19A, in certainembodiments, some embodiments of synthesizers of the present inventioncomprise a top enclosure (e.g. 97) that forms a primarily enclosed space104 over a top cover (e.g., 30, not shown in this figure). In preferredembodiments, the top enclosure has four sides (e.g., 98, two of whichare shown in FIG. 19A), and a top panel (e.g., 99) that form a primarilyenclosed space 104 above the top cover (e.g., 30) containing a pluralityof valves (e.g., 10, not shown in this figure) and a plurality ofdispense lines (e.g., 6, not shown in this figure). In certainembodiments, the top panel (e.g., 99) contains an outer window (e.g.,101). In some preferred embodiments, the outer window contains aventilation opening (e.g., 105).

As used herein, the combination of a top enclosure (e.g., 97) and topcover (e.g., 30) is referred to collectively as the “lid enclosure”(e.g., 102). In preferred embodiments, the “lid enclosure” has sixsides, with the top cover (e.g., 30) serving as the “bottom”, the toppanel serving as the surface opposite the top cover, and the four sidewalls being the top enclosure sides (e.g., 98). In certain embodiments,the lid enclosure has a ventilation opening (e.g., 105) with aventilation tube (e.g., 103) attached thereto (See, FIG. 19B). Inpreferred embodiments, the ventilation tube is connected to aventilation opening in an outer window 101.

In other embodiments, the synthesizer base (e.g., 2) comprises aprimarily enclosed space 104. In certain embodiments, a base (e.g., 2)of a synthesizer comprises a ventilation opening (e.g., 105) with aventilation tube (e.g., 103) attached thereto (See, e.g., FIGS. 22A and22B).

The ventilation openings in the lid enclosure or the base may be in anysuitable position. For example, the ventilation opening in the lidenclosure may be in the top panel (e.g. in the center, toward the backof the machine, or in one of the corners). The ventilation opening mayalso be located in a top enclosure side. For example, the ventilationopening may be in the enclosure side at the back of the machine, or onone of the sides (e.g., configured such that the lid enclosure may stillbe moved upward and downward while attached to a ventilation tube). Aventilation opening in a base may be, for example, on the front, thesides or on the back (e.g., configured such that the lid enclosure maystill be moved upward and downward without interference by theventilation tube). In preferred embodiments, the ventilation opening ispositioned toward the rear (e.g., on a side or in the back) to allow theventilation tubing to be directed away from an instrument operator. Inparticularly preferred embodiments, the ventilation opening is on theback of the base, e.g., as shown in FIGS. 22A and 22B.

In some embodiments, the ventilation is located in a position such thatair traveling through the primarily enclosed space (e.g., 104) makegreater or less contact with particular synthesizer components locatedinside the lid enclosure (e.g. valves, solenoids, dispense lines, etc.).The lid enclosures of the present invention may also have a plurality ofventilation openings. This may be desirable in order to control ordirect air flow through the primarily enclosed space (e.g., to minimizeor to maximize air contact with particular synthesizer components insidethe lid enclosure).

As shown in FIG. 19C, in certain embodiments, the lid enclosure ishinged so that is may be moved upward and downward (e.g., allowingaccess to the chamber bowl or other reaction chamber by a user). In someembodiments, the primarily enclosed space of the lid enclosure (e.g.104, not shown in this figure) is open to the ambient environmentthrough a ventilation slot (e.g. 100) in the top cover or the topenclosure (e.g. in top enclosure side wall towards the back of themachine).

In certain embodiments of the present invention, a lid enclosure ispresent on a commercially available machine (e.g., ABI 3900), and thelid enclosure is modified as described herein (e.g., a ventilationopening is made in the lid enclosure) An opening near the hinge forwiring serves as a ventilation slot on the 3900. In other embodiments,the lid enclosure must be added to synthesizer. For example, asynthesizer that simply has a top cover (e.g., 30), may have a topenclosure (e.g., 97) added thereto. This may be done by attaching a topenclosure that has bottom flanges (opposite the top panel) that fitaround the top cover, and provide a point of attachment (e.g., bolts,screws, adhesives, etc.). In other embodiments, the lid enclosure isfabricated as a separate component, then installed onto a synthesizer.For example, the components making up the lid enclosure (top enclosureand top cover) may be formed from a single mold, or two molds, etc. Inthis regard, features of the present invention may be built into the lidenclosure, such as the ventilation opening, ventilation slot, andcertain hood components (described below).

In some embodiments, e.g., as diagrammed in FIGS. 19A-C, the lidenclosure (e.g., 102) comprises, or is modified to comprise at least oneventilation opening (e.g., 105). One or more ventilation openings may beused. In preferred embodiments, a ventilation opening is placed in thecenter of the top panel so as to avoid blocking the operator's view ofinternal components, such as the synthesis columns, during operation. Inpreferred embodiments, the lid enclosure comprises windows constructedof transparent or translucent material, such as plexiglass.

In preferred embodiments, the lid enclosures of the present inventioncomprise a top panel directly opposite a top cover, and side wallsbetween these two components The primarily enclosed space between thetop panel and top cover is, in some embodiments, open to the ambientenvironment through a ventilation slot near the lid enclosure hinge(e.g., 106). In certain embodiments, the lid enclosure of the presentinvention comprises an inner window and an outer window (e.g. an outerwindow in the top panel, and an inner window in the top cover). Theouter window of the instrument allows visual inspection of operationsand components within the lid and within the chamber bowl 18 of the base2. The inner window seals the chamber bowl 18 by pressing against thechamber gasket when the lid enclosure is closed. Reagent supply tubingpasses through the inner window, but the window is sealed around eachtube so that the chamber will maintain appropriate pressure duringoperation. In the embodiment shown in FIGS. AB and 19B, the ventilationopening provides an aperture is the outer window.

In preferred embodiments, the ventilation opening (e.g., 105) isattached to a ventilation tube (e.g., 103), that in turn may be attachedto an exhaust system. In some embodiments, a synthesizer is attached toan individual exhaust system. In other embodiments, multiplesynthesizers are attached to a centralized exhaust system (e.g.centralized venting or vacuum system). In a preferred configuration,access to the exhaust system is toward the rear of the instrument, tominimize or prevent interference by the ventilation tubing with operatoraccess to the chamber bowl, and to conduct the fumes away frominstrument operators. The centralized exhaust may be a constant vacuumor a periodically actuated vacuum. In particular embodiments, raisingthe top cover or lid enclosure of a synthesizer triggers the vacuumsystem. In certain embodiments, reagent bottles on the sides of asynthesizer may also be vented through ventilation ports employing thesame ventilation system employed by the ventilation tube attached to thetop panel.

Another aspect of the present invention is to provide a ventilatedworkspace (e.g., around the chamber bowl) having a negative air pressurerelative to the surrounding air pressure, such that the flow of air goesfrom the surrounding room into the ventilated workspace, and not in thereverse, during operation of the ventilation system (e.g., as shown inFIGS. 21B and 22B). The ventilated workspace is designed to allow theinstrument operator to reach into the space (e.g., to remove thesynthesis columns) without turning off the ventilation system. Oneembodiment of a ventilated workspace is shown in FIG. 20A, wherein theventilated workspace is created by providing side panels (e.g., 107).Two variations of another embodiment are shown in FIGS. 20B and 20C. Inthis embodiment, the ventilated workspace is created by providing sidepanels (e.g., 107) between the body of the synthesizer and the lidenclosure, and a front panel (e.g., 108). In certain embodiments, theventilated workspace is created by including only side panels. In otherembodiments, the ventilated workspace is created by only including afront panel. In preferred embodiments, side and front panels are usedtogether (e.g., as in FIGS. 20B and 20C) to create a ventilatedworkspace. In some embodiments, side and front panels are provided asseparate components. In other embodiments, a single component comprisingboth side panels and a front panel is provided.

The size of the ventilated workspace can be altered by the placement ofthe panels, e.g., the side panels (107) shown in FIGS. 20 A-C. In someembodiments, panels are positioned to maximize the size of the enclosedventilated workspace (e.g., as in FIG. 20B). In other embodiments, thepanels are positioned to provide a smaller ventilated workspace (e.g.,as with the side panels in FIG. 20C). In some preferred embodiments, theside panels are positioned as close to the top chamber gasket (e.g., 31)as they can be without disturbing the seal between the top chambergasket and the top cover 30. In certain embodiments, the front and/orside panels are used with a synthesizer only having a top cover (not afull lid enclosure).

The side panels can be made of a number of different materials. In someembodiments, the materials used for the side panels are opaque. In otherembodiments, the side panels are translucent or clear (e.g., to permitsurrounding light into the ventilated workspace). In certainembodiments, the side panels are constructed from flexible polymericmaterial (e.g., sheeting), such as polyethylene or polypropylene. Insome embodiments, the polymeric material has an average thickness ofabout 2 to 8 mils. In preferred embodiments, the polymeric material hasan average thickness of about 2 to 4 mils. In some embodiments, thepanels are collapsible (i.e., can collapse or fold down upon themselvesas the lid enclosure or top cover, is lowered). In some embodiments,panels are accordion-style or fan-fold style barriers that fold downupon themselves when the top cover or lid enclosure is lowered. Inpreferred embodiments, when the panels are collapsed, they have a totalthickness that is less than the height of the O-ring or gasket (e.g.,top chamber seal 31) on the interior of the synthesizer (e.g., so thatthere is no interference with the sealing of the O-ring).

In other embodiments, the side panels are constructed of rigid material.In some embodiments, rigid side panels are configured to fit intorecesses in the body of the synthesizer when the top cover or lidenclosure is closed. In other embodiments, rigid side panels areconfigured to fit come down around the outside of the base of thesynthesizer when the top cover or lid enclosure is closed. In someembodiments, rigid side panels are constructed from opaque materials(e.g., steel, aluminum, opaque plastic). In other embodiments, rigidside panels are constructed from translucent or transparent material,such as plexiglass. Generally, the side panels are connected to the topcover, so when the top cover or lid enclosure is raised, the side panelsslide up to form sides for the ventilated workspace.

In certain embodiments, a front panel (e.g., 108) is attached to the lidenclosure. For example, the front panel may attach to the top cover(e.g., FIG. 20B), or the front panel may attach to one of sides of thelid enclosure (e.g., FIG. 20C). The front panel may drape over the frontof the synthesizer when the lid enclosure is closed (See, e.g., FIGS.19B and 20C). Alternatively, the front panel may fit into a recessedslot in the synthesizer base, or fold up upon itself as the lidenclosure is lowered into the closed position.

Attachment of the panels provided for the purpose of enclosing theventilated workspace is not limited to any particular means. Forexample, in a simple configuration, panels are attached by use of stripsof VELCRO fastener (e.g., adhesive backed strips), for easy mounting andremoval. For a sturdier attachment, the panels may be attached usingfasteners, including but not limited to screws, bolts, welds, and snaps,or may be attached with removable or permanent adhesives. The presenceof the panels reduces the size of the opening through which ambient aircan enter the ventilated workspace, and also reduces the size of theopening from which air and vapors in the chamber bowl can escape. Whenthe ventilation system is turned on (e.g., when the connectedventilation tube is drawing air from the ventilation opening, theairflow through the reduced opening prevents or reduces any flow (e.g.outward flow) of gaseous emissions. When the ventilation system isactuated, ambient air and reagent vapors are drawn across the chamberbowl (e.g., 18) and into the ventilation slot (e.g., 100), as diagrammedin FIGS. 21B and 22B. The air and vapors then move through the primarilyenclosed space (e.g., 104) and exit through the ventilation opening(e.g., 105) into the ventilation tube (e.g., 103). In some embodiments,the air flow rate at the opening of the ventilated workspace (e.g., inthe embodiments shown in FIGS. 20B and 20B, where the surrounding air isdrawn into the ventilated workspace below the front panel and betweenthe side panels) is from about 20 to about 100 feet per minute, facevelocity. In some preferred embodiments, the flow rate at the opening isabout 40 to 50 feet per minute, face velocity.

From the ventilation tube, the air and vapors may be vented, treated orcollected. In certain embodiments, the vented air and vapors are routedto a central scrubber. The central scrubber may form part of an overallemission control system. The central system may also be used to adjusttotal airflow for the number of synthesizers that are open at the sametime. In this regard, exhaust from the system is minimized so as toconcentrate waste vapors.

In order to increase or decrease the speed at which air and vaporstravels through the ventilation system of the present invention, thesize of the ventilation slot may be adjusted (e.g. reducing the size ofthe ventilation slot increase the speed of the moving air and vapors).The airflow pattern made possible by the present invention allowssynthesizers to be opened (e.g. to change columns, etc) without exposureof an operator to hazardous vapors (e.g. argon, solvent fumes, etc).

The integrated chamber ventilation system of the present invention maybe adapted to many synthesizers of both ‘open’ and ‘closed’ design. Onexample of another synthesizer that can be modified to include thereaction enclosure ventilation system of the present invention is thePOLYPLEX 96-channel, high-throughput oligonucleotide synthesizer fromGeneMachines, San Carlos, Calif., which comprises a synthesis caseproviding an enclosure for the synthesis block in which the reactionsare performed. A similar instrument is described in WO 00/56445,published Sep. 28, 2000, and in related U.S. Provisional Patentapplication 60/125,262, filed Mar. 19, 1999, each incorporated herein intheir entireties. As described in WO 00/56445, the synthesis case has aloading station, drain station, and water-tolerant and water-sensitivereagent filling stations. The synthesis case has a cover, a first and asecond side, a first and a second end, and a bottom side, which contactsthe base. The load station comprises a sealable opening in the synthesiscase through which a multiwell plate can be inserted. In application ofthe present invention, the synthesis case can be fitted with one or moreventilation openings similar to ventilation opening 105, for attachmentto ventilation tubing (e.g., 103). In some embodiments, a ventilationopening is in a side of the synthesis case opposite the side having thesealable opening. In preferred embodiments, a ventilation opening in thesynthesis case is on the first or second end. In particularly preferredembodiments, the ventilation system is actuated when the sealableopening is opened, e.g., for insertion or removal of a multiwell plate.

II) Production Facilities

The present invention provides synthesizer arrays (e.g., groups ofsynthesizers). In some embodiments, the synthesizers are arranged inbanks. For example, a given bank of synthesizers may be used to produceone set of oligonucleotides. The present invention is not limited to anyone synthesizer. Indeed, a variety of synthesizers are contemplated,including, but not limited to the synthesizers of the present invention,MOSS EXPEDITE 16-channel DNA synthesizers (PE Biosystems, Foster City,Calif.), OligoPilot (Amersham Pharmacia,), and the 3900 and 394848-Channel DNA synthesizers (PE Biosystems, Foster City, Calif.). Insome embodiments, synthesizers are modified or are wholly fabricated tomeet physical or performance specifications particularly preferred foruse in the synthesis component of the present invention. In someembodiments, two or more different DNA synthesizers are combined in onebank in order to optimize the quantities of different oligonucleotidesneeded. This allows for the rapid synthesis (e.g., in less than 4 hours)of an entire set of oligonucleotides (all the oligonucleotide componentsneeded for a particular assay, e.g., for detection of one SNP using anINVADER assay [Third Wave Technologies, Madison, Wis.]).

In some embodiments the DNA synthesizer component includes at least 100synthesizers. In other embodiments, the DNA synthesizer componentincludes at least 200 synthesizers. In still other embodiments, the DNAsynthesizer component includes at least 250 synthesizers. In someembodiments, the DNA synthesizers are run 24 hours a day.

A. Automated and Fail-Safe Reagent Supply

In some embodiments, the DNA synthesizers in the oligonucleotidesynthesis component further comprise an automated reagent supply system.The automated reagent supply system delivers reagents necessary forsynthesis to the synthesizers from a central supply area. In someembodiments, the central supply area is provided in an isolated roomequipped for accommodating leakage, fires, and explosions withoutthreatening other portions of the synthesis facility, the environment,or humans. Where the central supply area provides reagents for multiplesynthesizers, in some embodiments, the system is configured to allowbanks of synthesizer or individual synthesizer to be removed from thesystem (e.g., for maintenance or repair) without interrupting activityat other synthesizers. Thus, the present invention provides an efficientfail-safe reagent delivery system.

For example, in some embodiments, acetonitrile is supplied via tubing(e.g., stainless steel or TEFLON tubing) through the automated supplysystem. De-blocking solution may also be supplied directly to DNAsynthesizers through tubing. In some preferred embodiments, the reagentsupply system tubing is designed to connect directly to the DNAsynthesizers without modifying the synthesizers. Additionally, in someembodiments, the central reagent supply is designed to deliver reagentsat a constant and controlled pressure. The amount of reagent circulatingin the central supply loop is maintained at 8 to 12 times the levelneeded for synthesis in order to allow standardized pressure at eachinstrument. The excess reagent also allows new reagent to be added tothe system without shutting down. In addition, the excess of reagentallows different types of pressurized reagent containers to be attachedto one system. The excess of reagents in one centralized system furtherallows for one central system for chemical spills and fire suppression.

In some embodiments, the DNA synthesis component includes a centralizedargon delivery system. The system includes high-pressure argon tanksadjacent to each bank of synthesizers. These tanks are connected tolarge, main argon tanks for backup. In some embodiments, the main tanksare run in series. In other embodiments, the main tanks are set up inbanks. In some embodiments, the system further includes an automatedtank switching system. In some preferred embodiments, the argon deliverysystem further comprises a tertiary backup system to provide argon inthe case of failure of the primary and backup systems.

In some embodiments, one or more branched delivery components are usedbetween the reagent tanks and the individual synthesizers or banks ofsynthesizers. For example, in some embodiments, acetonitrile isdelivered through a branched metal structure (e.g., the structuredescribed in FIG. 14). Where more than one branched delivery componentis used, in preferred embodiments, each branched delivery component isindividually pressurized.

The present invention is not limited by the number of branches in thebranched delivery component. In preferred embodiments, each brancheddelivery component (76) contains ten or more branches (77). Reagenttanks may be connected to the branched delivery components using anynumber of configurations. For example, in some embodiments, a singlereagent tank is matched with a single branched component. In otherembodiments, a plurality of reagent tanks is used to supply reagents toone or more branched components. In some such embodiments, the pluralityof tanks may be attached to the branched components through a singlefeed line, wherein one or a subset of the tanks feeds the branchedcomponents until empty (or substantially empty), whereby a second tankor subset of tanks is accessed to maintain a continuous supply ofreagent to the one or more branched components. To automate themonitoring and switching of tanks, an ultrasonic level sensor may beapplied.

In some embodiments, each branch of the branched delivery componentprovides reagent to one synthesizer or to a bank of synthesizers throughconnecting tubing (78). In preferred embodiments, tubing is continuous(i.e., provides a direct connection between the delivery branch and thesynthesizer). In some preferred embodiments, the tubing comprises aninterior diameter of 0.25 inches or less (e.g., 0.125 inches). In someembodiments, each branch contains one or more valves (preferably one).While the valve may be located at any position along the delivery line,in preferred embodiments, the valve is located in close proximity to thesynthesizer. In other embodiments, reagent is provided directly tosynthesizers without any joints or valves between the branched deliverycomponent and the synthesizers.

In some embodiments, the solvent is contained in a cabinet designed forthe safe storage of flammable chemicals (a “flammables cabinet”) and thebranched structure is located outside of the cabinet and is fed by thesolvent container through tubing passed through the wall of the cabinet.In other embodiments, the reagent and branched system is stored in anexplosion proof room or chamber and the solvent is pumped via tubingthrough the wall of the explosion proof room. In preferred embodiments,all of the tubing from each of the branches is fed through the wall inat a single location (e.g., through a single hole (79) in the wall(80)).

The reagent delivery system of the present invention provides severaladvantages. For example, such a system allows each synthesizer to beturned off (e.g., for servicing) independent of the other synthesizers.Use of continuous tubing reduces the number of joints and couplings, theareas most vulnerable to failure, between the reagent sources and thesynthesizers, thereby reducing the potential for leakage or blockage inthe system. Use of continuous tubing through inaccessible ordifficult-to-access areas reduces the likelihood that repairs or servicewill be needed in such areas. In addition, fewer valves results in costsavings.

In some embodiments, the branched tubing structure further provides asight glass (81). In preferred embodiments, the sight glass is locatedat the top of the branched delivery structure. The sight glass providesthe opportunity for visual and physical sampling of the reagent. Forexample, in some embodiments, the sight glass includes a sampling valve(82) (e.g., to collect samples for quality control). In someembodiments, the site glass serves as a trap for gas bubbles, to preventbubbles from entering the connecting tubing (78). In other embodiments,the sight glass contains a vent (e.g., a solenoid valve) for de-gassingof the system (83). In some embodiments, scanning of the sight glass(e.g., spectrophotometrically) and sampling are automated. The automatedsystem provides quality control and feedback (e.g., the presence ofcontamination).

In other embodiments, the present invention provides a portable reagentdelivery system. In some embodiments, the portable reagent deliverysystem comprises a branched structure connected to solvent tanks thatare contained in a flammables cabinet. In preferred embodiments, onereagent delivery system is able to provide sufficient reagent for 40 ormore synthesizers. These portable reagent delivery systems of thepresent invention facilitate the operation of mobile (portable)synthesis facilities. In another embodiment, these portable reagentdelivery systems facilitate the operation of flexible synthesisfacilities that can be easily re-configured to meet particular needs ofindividual synthesis projects or contracts. In some embodiments, asynthesis facility comprises multiple portable reagent delivery systems.

B. Waste Collection

In some embodiments, the DNA synthesis component further comprises acentralized waste collection system. The centralized waste collectionsystem comprises cache pots for central waste collection. In someembodiments, the cache pots include level detectors such that when wastelevel reaches a preset value, a pump is activated to drain the cacheinto a central collection reservoir. In preferred embodiments, ductworkis provided to gather fumes from cache pots. The fumes are then ventedsafely through the roof, avoiding exposure of personnel to harmfulfumes. In preferred embodiments, the air handling system provides anadequate amount of air exchange per person to ensure that personnel arenot exposed to harmful fumes. The coordinated reagent delivery and wasteremoval systems increase the safety and health of workers, as well asimproving cost savings.

In some embodiments, the solvent waste disposal system comprises a wastetransfer system. In some preferred embodiments, the system contains noelectronic components. In some preferred embodiments, the systemcomprises no moving parts. For example, in some embodiments, waste isfirst collected in a liquid transfer drum (84) designed for the safestorage of flammable waste (See FIG. 15 for an exemplary waste disposalsystem). In some embodiments, waste is manually poured into the drumthrough a waste channel (85). In preferred embodiments, solvent waste isautomatically transported (e.g., through tubing) directly fromsynthesizers to the drum (84). To drain the liquid transfer drum (84),argon is pumped from a pressurized gas line (86) into the drum through afirst opening (87), forcing solvent waste out an output channel (88) ata second opening (89) (e.g., through tubing) into a centralized wastecollection area. In preferred embodiments, the argon is pumped at lowpressure (e.g., 3-10 pounds per square inch (psi), preferably 5 psi orless). In some embodiments, the drum (84) contains a sight glass (90) tovisualize the solvent level. In some embodiments, the level isvisualized manually and the disposal system is activated when the drum(84) has reached a selected threshold level (91). In other embodiments,the level is automatically detected and the disposal system isautomatically activated when the drum (84) has reached the thresholdlevel (91).

The solvent waste transfer system of the present invention providesseveral advantages over manual collection and complex systems. Thesolvent waste system of the present invention is intrinsically safe, asit can be designed with no moving or electrical parts. For example, thesystem described above is suitable for use in Division I/Class I spaceunder EPA regulations.

Some process steps may put out caustic waste. For example, deprotectionof synthesized oligonucleotides generally includes treatment with NH₄OH.In some embodiments, caustic waste is neutralized before disposal, e.g.,to a sanitary sewer. In preferred embodiments, the neutralization of thewaste is checked (e.g., by measurement of pH) to ensure that it is in anappropriate condition for disposal via the intended system (e.g., thesanitary sewer system).

In some embodiments, waste from each deprotection station is neutralizedbefore collection to a centralized waste collection or disposal system.In other embodiments, caustic waste from a plurality of deprotectionstations is collected before neutralization.

By way of example, and not intended as a limitation, the followingprovides a description for one embodiment of a centralized collectionand neutralization system for caustic waste. The system may comprisecollection of caustic waste from one or more stations in a tank, e.g., acarboy. In some embodiments, the amount of neutralizing reagent requiredto neutralize a defined amount of caustic waste is calculated, based onthe volume and content of the waste. In some embodiments, the calculatedamount of neutralizing reagent is added after collection of the waste.In preferred embodiments, the calculated amount of neutralizing reagentis provided in the carboy, such that when the carboy is full or when thecombined volume of the neutralizer and waste reaches a predeterminedvolume, the waste has been neutralized.

In one embodiment, the carboy is provided with a pH probe formeasurement of the pH of the collected waste. In some embodiments, thesystem provides a means of altering the pH of the collected waste. Inpreferred embodiments, the altering of the pH occurs in response to ameasured pH value for the collected waste. For example, if the pH isdetermined to be outside a certain range, (e.g., if it does not fallbetween, for example, pH 7 and pH 9), the system provides a reagentselected to adjust the pH to the selected range (e.g., if the pH isfound to be high, the system dispenses an acidic solution forneutralization; if the pH is low, the system dispenses a basic solutionfor neutralization). When the pH comes into the selected range, thesystem shuts off the dispenser. For the step of dispensing aneutralizing reagent, any system suitable for the controlled delivery ofa reagent is contemplated. For example, discharge may be accomplishedvia a mechanical dispenser, or discharge can be accomplished vianon-mechanical means, e.g., via control of air pressure.

In some embodiments, neutralization treatment is provided to thecollected waste in bulk, e.g., when the carboy is full or when itreaches a predetermined threshold level. In other embodiments,neutralization is periodic. In some embodiments, periodic neutralizationis set to occur at particular times, e.g., at particular times of day,or whenever a particular interval of time has passed since the lasttreatment. In other embodiments, periodic treatment is set to respond toa condition of the waste container, such as whenever a new addition ofwaste material occurs, or whenever the pH is not within the selectedrange. In yet other embodiments, periodic treatment occurs based on acombination of these or other factors.

In a preferred embodiment, the carboy is provided with a means formixing, such as a stirrer or agitator. In some embodiments, the systemcomprises a device for keeping a precipitate suspended. In someembodiments, the system provides a filter for removing precipitates,particulates or other non-liquid matter in the collected waste. In otherpreferred embodiments, the system provides a means of venting gasses. Inparticularly preferred embodiments, the gasses are collected fordisposal through a centralized ventilation system.

C. Centralized Control System

In some embodiments, all of the DNA synthesizers in the synthesiscomponent are attached to a centralized control system. The centralizedcontrol system controls all areas of operation, including, but notlimited to, power, pressure, reagent delivery, waste, and synthesis. Insome preferred embodiments, the centralized control system includes aclean electrical grid with uninterrupted power supply. Such a systemminimizes power level fluctuations. In additional preferred embodiments,the centralized control system includes alarms for air flow, status ofreagents, and status of waste containers. The alarm system can bemonitored from the central control panel. The centralized control systemallows additions, deletions, or shutdowns of one synthesizer or oneblock of synthesizers without disrupting operations of otherinstruments. The centralized power control allows user to turninstruments off instrument-by-instrument, bank-by-bank, or the entiremodule.

D. Integrated Production Process

In some embodiments, the present invention provides an automatedproduction process. In some embodiments, the automated productionprocess includes an oligonucleotide synthesizer component and anoligonucleotide-processing component. In some embodiments, theoligonucleotide production component includes multiple components,including but not limited to, an oligonucleotide cleavage anddeprotection component, an oligonucleotide purification component, anoligonucleotide dry down component; an oligonucleotide de-saltingcomponent, an oligonucleotide dilute and fill component, and a qualitycontrol component. In some embodiments, the automated DNA productionprocess of the present invention further includes automated designsoftware and supporting computer terminals and connections, a producttracking system (e.g., a bar code system), and a centralized packagingcomponent. In some embodiments, the components are combined in anintegrated, centrally controlled, automated production system. Thepresent invention thus provides methods of synthesizing several relatedoligonucleotides (e.g., components of a kit) in a coordinated manner. Insome embodiments, a sample holder (e.g., a reaction support) is sharedbetween two or more of the components of the production process. Thesample holder may be transferred by hand or robotically from onecomponent to the next.

1. Oligonucleotide Design Component

In some embodiments of the present invention, the DNA production processincluded an automated oligonucleotide design system. The system includessoftware utilized to design the sequence of the oligonucleotide. Thesoftware and parameters chosen vary according to the application thatthe oligonucleotides are designed for use in.

For example, in some embodiments where an oligonucleotide is designedfor use in the INVADER assay to detect a SNP, the sequence(s) ofinterest (synthesis request information) are entered into theINVADERCREATOR program (Third Wave Technologies, Madison, Wis.). Theprogram designs probes for both the sense and antisense strand. Strandselection is based upon the ease of synthesis, minimization of secondarystructure formation, and manufacturability. In some embodiments, theuser chooses the strand for sequences to be designed for. In otherembodiments, the software automatically selects the strand. Byincorporating thermodynamic parameters for optimum probe cycling andsignal generation (Allawi and SantaLucia, Biochemistry, 36:10581[1997]), oligonucleotide probes are designed to operate at a preselectedassay temperature. In particular embodiments, oligonucleotide probes aredesigned to operate at an assay temperature of 63° C. Based on thesecriteria, a final probe set (e.g., primary probes for 2 alleles and anINVADER oligonucleotide) is selected.

In some embodiments, the INVADERCREATOR system is a web-based programwith secure site access that contains a link to the BLAST search website at the National Library of Medicine at the NIH, and can be linkedto RNAstructure (Mathews et al., RNA 5:1458[1999]), a software programthat incorporates mfold (Zuker, Science, 244:48[1989]). RNAstructuretests the proposed oligonucleotide designs generated by INVADERCREATORfor potential uni- and bimolecular complex formation. INVADERCREATOR isopen database connectivity (ODBC)-compliant and uses the Oracle databasefor export/integration. The INVADERCREATOR system was configured withOracle to work well with UNIX systems, as most genome centers areUNIX-based.

In preferred embodiments, the INVADERCREATOR analysis is provided on aseparate Sun server so it can handle analysis of large batch jobs. Forexample, a customer can submit up to 2,000 SNP sequences in one email.The server passes the batch of sequences on to the INVADERCREATORsoftware, and, when initiated, the program designs SNP sets. Probe setdesigns are returned to the user within 24 hours of receipt of thesequences.

Each INVADER reaction includes at least two target sequence-specificoligonucleotides for the primary reaction: an upstream INVADERoligonucleotide and a downstream Probe oligonucleotide. Generally, theseoligonucleotides are unlabeled. The INVADER oligonucleotide is designedto bind stably at the reaction temperature, while the probe is designedto freely associate and disassociate with the target strand, withcleavage occurring only when an uncut probe hybridizes to a targetadjacent to an overlapping INVADER oligonucleotide. In some embodiments,the probe includes a 5′ flap that is not complementary to the target,and this flap is released from the probe when cleavage occurs. In someembodiments, the released flap participates as an INVADERoligonucleotide in a secondary reaction.

To select a probe sequence that will perform optimally at a pre-selectedreaction temperature, the melting temperature (TM) of the SNP to bedetected is calculated using the nearest-neighbor model and publishedparameters for DNA duplex formation (Allawi and SantaLucia,Biochemistry, 36:10581 [1997]. Because the assay's salt concentrationsare often different than the solution conditions in which thenearest-neighbor parameters were obtained (1M NaCl and no divalentmetals), and because the presence and concentration of the enzymeinfluences the optimal reaction temperature, an adjustment is generallymade to the calculated TM to determine the optimal temperature at whichto perform a reaction. One way of compensating for these factors is tovary the value provided for the salt concentration within the meltingtemperature calculations. This adjustment is termed a ‘salt correction’.As used herein, the term “salt correction” refers to a variation made inthe value provided for a salt concentration for the purpose ofreflecting the effect on a TM calculation for a nucleic acid duplex of anon-salt parameter or condition affecting said duplex. Variation of thevalues provided for the strand concentrations will also affect theoutcome of these calculations. By using a value of 0.5 M NaCl(SantaLucia, Proc Natl Acad Sci USA, 95:1460[1998]) and strandconcentrations of about 1 mM of the probe and 1 fM target, the algorithmused for calculating probe-target melting temperature has been adaptedfor use in predicting optimal INVADER assay reaction temperature. For aset of 30 probes, the average deviation between optimal assaytemperatures calculated by this method and those experimentallydetermined is about 1.5° C.

The length of the downstream probe to a given SNP is defined by thetemperature selected for running the reaction (e.g., 63° C.). Startingfrom the position of the variant nucleotide on the target DNA (thetarget base that is paired to the probe nucleotide 5′ of the intendedcleavage site), an iterative procedure is used by which the length ofthe SNP region is increased by one base pair until a calculated optimalreaction temperature (TM plus salt correction to compensate for enzymeeffect) matching the pre-selected, desired reaction temperature isreached. The non-complementary arm of the probe is preferably selectedto allow the secondary reaction to cycle at the same reactiontemperature, and is screened using programs such as mfold (Zuker,Science, 244: 48 [1989]) or Oligo 5.0 (Rychlik and Rhoads, Nucleic AcidsRes, 17: 8543 [1989]) for the possible formation of dimer complexes orsecondary structures that could interfere with the reaction. The sameprinciples are also followed for INVADER oligonucleotide design.Briefly, starting from the position N on the target DNA, the 3′ end ofthe INVADER oligonucleotide is designed to have a nucleotide notcomplementary to either allele suspected of being contained in thesample to be tested. The mismatch does not adversely affect cleavage(Lyamichev et al., Nature Biotechnology, 17: 292 [1999]), and it canenhance probe cycling, presumably by minimizing coaxial stabilizationeffects between the two probes. Additional residues complementary to thetarget DNA starting from residue N−1 are then added in the upstreamdirection until the stability of the INVADER oligonucleotide-targethybrid exceeds that of the probe (and therefore the planned assayreaction temperature) by 15-20° C.

It is one aspect of the assay design that the all of the probe sequencesmay be selected to allow the primary and secondary reactions to occur atthe same optimal temperature, so that the reaction steps can runsimultaneously. In an alternative embodiment, the probes may be designedto operate at different optimal temperatures, so that the reactionssteps are not simultaneously at their temperature optima.

The present invention is not limited to the use of the INVADERCREATORsoftware. Indeed, a variety of software programs are contemplated andare commercially available, including, but not limited to PRIMER EXPRESS(Applied Biosystems, Foster City, Calif.), GCG Wisconsin Package(Genetics computer Group, Madison, Wis.) and Vector NTI (Informax,Rockville, Md.).

2. Oligonucleotide Synthesis Component

Once a particular oligonucleotide sequence or set of sequences has beenchosen, sequences are sent (e.g., electronically) to a high-throughputoligonucleotide synthesizer component. In some preferred embodiments,the high-throughput synthesizer component contains multiple DNAsynthesizers. Such systems are described in detail above.

3. Oligonucleotide Processing Components

In some embodiments, the automated DNA production process furthercomprises one or more oligonucleotide production components, including,but not limited to, an oligonucleotide cleavage and deprotectioncomponent, an oligonucleotide purification component, a dry-downcomponent, a desalting component, a dilution and fill component, and aquality control component.

A. Oligonucleotide Cleavage and Deprotection

After synthesis is complete, the oligonucleotide synthesis columns aremoved to the cleavage and deprotection station. In some embodiments, thetransfer of oligonucleotides to this station is automated and controlledby robotic automation. In some embodiments, the entire cleavage anddeprotection process is performed by robotic automation. In someembodiments, a deprotecting reagent (e.g., NH₄OH or other deprotectingreagent) is supplied through the automated reagent supply system.Accordingly, in some embodiments, oligonucleotide deprotection isperformed in multi-sample containers (e.g., 96 well covered dishes) inan oven. This method is designed for the high-throughput system of thepresent invention and is capable of the simultaneous processing of largenumbers of samples. This method provides several advantages over thestandard method of deprotection in vials. For example, sample handlingis reduced (e.g., labeling of vials dispensing of concentrated NH₄OH toindividual vials, as well as the associated capping and uncapping of thevials, is eliminated). This reduces the risks of contamination ormislabeling and decreases processing time. Where such methods are usedto replace human pipetting of samples and capping of vials, the methodssave many labor hours per day. The method also reduces consumablerequirements by eliminating the need for vials and pipette tips, reducesequipment needs by eliminating the need for pipettes, and improvesworker safety conditions by reducing worker exposure to ammoniumhydroxide. The potential for repetitive motion disorders is alsoreduced. Deprotection in a multi-well plate further has the advantagethat the plate can be directly placed on an automated desaltingapparatus (e.g., TECAN Robot).

During the development of the present invention, the plate was optimizedto be functional and compatible with the deprotection methods. In someembodiments, the plate is designed to be able to hold as much as twomilliliters of oligonucleotide and ammonium hydroxide. If deep wellplates are used, automated downstream processing steps may need to bealtered to ensure that the full volume of sample is extracted from thewells. In some embodiments, the multi-well plates used in the methods ofthe present invention comprise a tight sealing lid/cover to protect fromevaporation, provide for even heating, and are able to withstandtemperatures and pressures necessary for deprotection. Attempts withinitial plates were not successful, having problems with lids that werenot suitably sealed and plates that did not withstand deprotectiontemperatures.

In some embodiments (e.g., processing of target and INVADERoligonucleotides), oligonucleotides are cleaved from the synthesissupport in the multi-well plates. In other embodiments (e.g., processingof probe oligonucleotides), oligonucleotides are first cleaved from thesynthesis column and then transferred to the plate for deprotection.

B. Oligonucleotide Purification

In some embodiments, following deprotection and cleavage from the solidsupport, oligonucleotides are further purified. Any suitablepurification method may be employed, including, but not limited to, highpressure liquid chromatography (HPLC) (e.g., using reverse phase C18 andion exchange), reverse phase cartridge purification, and gelelectrophoresis. However, in preferred embodiments, purification iscarried out using ion exchange HPLC chromatography.

In some embodiments, multiple HPLC instruments are utilized, andintegrated into banks (e.g., banks of 8 HPLC instruments). Each bank isreferred to as an HPLC module. Each HPLC module consists of an automatedinjector (e.g., including, but not limited to, Leap Technologies 8-portinjector) connected to each bank of automated HPLC instruments (e.g.,including, but not limited to, Beckman-Coulter HPLC instruments). Theautomatic Leap injector can handle four 96-well plates of cleaved anddeprotected oligonucleotides at a time. The Leap injector automaticallyloads a sample onto each of the HPLCs in a given bank. The use of oneinjector with each bank of HPLC provides the advantage of reducing laborand allowing integrated processing of information.

In some embodiments, oligonucleotides are purified on an ion exchangecolumn using a salt gradient. Any suitable ion exchange functionality orsupport may be utilized, including but not limited to, Source 15 Q ionexchange resin (Pharmacia). Any suitable salt may be utilized forelution of oligonucleotides from the ion exchange column, including butnot limited to, sodium chloride, acetonitrile, and sodium perchlorate.However, in preferred embodiments, a gradient of sodium perchlorate inacetonitrile and sodium acetate is utilized.

In some embodiments, the gradient is run for a sufficient time course tocapture a broad range of sizes of oligonucleotides. For example, in someembodiments, the gradient is a 54 minute gradient carried out using themethod described in Tables 1 and 2. Table 1 describes an HPLC protocolfor the gradient. The time column represents the time of the operation.The module column represents the equipment that controls the operation.The function column represents the function that the HPLC is performing.The value column represents the value of the HPLC function at the timespecified in the time column. Table 2 describes the gradient used inHPLC purification. The column temperature is 65° C. Buffer A is 20 mMSodium Perchlorate, 20 mM Sodium Acetate, 10% Acetonitrile, pH 7.35.Buffer B is 600 mM Sodium Perchlorate, 20 mM Sodium Acetate, 10%Acetonitrile, pH 7.35.

In some embodiments, the gradient is shortened. In preferredembodiments, the gradient is shortened so that a particular gradientrange suitable for the elution of a particular oligonucleotide beingpurified is accomplished in a reduced amount of time. In other preferredembodiments, the gradient is shortened so that a particular gradientrange suitable for the elution of any oligonucleotide having a sizewithin a selected size range is accomplished in a reduced amount oftime. This latter embodiment provides the advantages that the workerperforming HPLC need not have foreknowledge of the size of anoligonucleotide within the selected size range, and the protocol neednot be altered for purification of any oligonucleotide having a sizewithin the range.

In a particularly preferred embodiment, the gradient is a 34 minutegradient described in the Tables 3 and 4. The parameters and buffercompositions are as described for Tables 1 and 2. Reducing the gradientto 34 minutes increases the capacity of synthesis per HPLC instrumentand reduces buffer usage by 50% compared to the 54 minute protocoldescribed above. The 34 minute HPLC method of the present invention hasthe further advantage of being optimized to be able to separateoligonucleotides of a length range of 23-39 nucleotides without anychanges in the protocol for the different lengths within the range.Previous methods required changes for every 2-3 nucleotide change inlength. In yet other embodiments, the gradient time is reduced evenfurther (e.g., to less than 30 minutes, preferably to less than 20minutes, and even more preferably, to less than 15 minutes). Anysuitable method may be utilized that meets the requirements of thepresent invention (e.g., able to purify a wide range of oligonucleotidelengths using the same protocol).

In some embodiments, separate sets of HPLC conditions, each selected topurify oligonucleotides within a different size range, may be provided(e.g., may be run on separate HPLCs or banks of HPLCs). Thus, in someembodiments of the present invention, a first bank of HPLCs areconfigured to purify oligonucleotides using a first set of purificationconditions (e.g., for 23-39 mers), while second and third banks are usedfor the shorter and longer oligonucleotides. Use of this system allowsfor automated purification without the need to change any parametersfrom purification to purification and decreases the time required foroligonucleotide production.

In some embodiments, the HPLC station is equipped with a central reagentsupply system. In some embodiments, the central reagent system includesan automated buffer preparation system. The automated buffer preparationsystem includes large vat carboys that receive pre-measured reagents andwater for centralized buffer preparation. The buffers (e.g., a high saltbuffer and a low salt buffer) are piped through a circulation loopdirectly from the central preparation area to the HPLCs. In someembodiments, the conductivity of the solution in the circulation loop ismonitored to verify correct content and adequate mixing. In addition, insome embodiments, circulation lines are fitted with venturis for staticmixing of the solutions as they are circulated through the piping loop.In still further embodiments, the circulation lines are fitted with 0.05μm filters for sterilization. In some preferred embodiments, the buffertanks contain from about 100 liters to about 500 liters of buffer. Theuse of large buffer tanks allows for a more consistent buffer mixture.In some preferred embodiments, the individual buffer systems aresupported by a high purity water purification system so as to avoidhaving to purchase individual containers.

In some preferred embodiments, the HPLC purification step is carried outin a clean room environment. The clean room includes a HEPA filtrationsystem. All personnel in the clean room are outfitted with protectivegloves, hair coverings, and foot coverings.

In preferred embodiments, the automated buffer prep system is located ina non-clean room environment and the prepared buffer is piped throughthe wall into the clean room.

Each purified oligonucleotide is collected into a tube (e.g., a 50-mlconical tube) in a carrying case in the fraction collector. Collectionis based on a set method, which is triggered by an absorbance ratechange, level, or threshold value within a predetermined time window. Insome embodiments, the method uses a flow rate of 5 ml/min (the maximumrate of the pumps is 10 ml/min.) and each column is automatically washedbefore the injector loads the next sample.

TABLE 1 54 Minute HPLC Method Time (min) Module Function Value Duration(min) 0 Pump % B 22.00 4.0 0 Det 166-3 Autozero ON 0 Det 166-3 Relay ON3.0 0.10 4 Pump % B 37.00 43.00 47 Pump % B 100.00 0.50 47.5 Pump FlowRate 7.5 0.00 50.0 Pump % B 5.0 0.50 53.45 Det 166-3 Stop Data (Det =detector; % B = percent of buffer B; flow rate values in ml/min)

TABLE 2 54 Minute HPLC Method Time Gradient Flow Rate 0 5% B/95% A 5ml/min 0-4 min 5-22% B 5 ml/min 4-47 min 22-37% B 5 ml/min 47-47.5 min37-100% B 7.5 ml/min 47.5-50 min 100% B 7.5 ml/min 50-50.5 min 100-5% B7.5 ml/min 50.5-53.5 min 5% B 7.5 ml/min

TABLE 3 34 Minute HPLC Method Time (min) Module Function Value Duration0 Pump % B 26.00 2.0 0 Det 166-3 Autozero ON 0 Det 166-3 Relay ON 3.00.10 2 Pump % B 36.00 27.00 29 Pump % B 100.00 0.50 29.5 Pump Flow Rate7.5 0.00 32 Pump % B 5.0 0.50 33.45 Det 166-3 Stop Data

TABLE 4 34 Minute HPLC Method Time Gradient Flow Rate 0 5% B/95% A 5ml/min 0-2 min 5-26% B 5 ml/min 2-29 min 26-36% B 5 ml/min 29-29.5 min36-100% B 6.5 ml/min 29.5-32 min 100% B 7.5 ml/min 32-32.5 min 100-5% B7.5 ml/min 32.5-33.5 min 5% B 7.5 ml/min

C. Dry-Down Component

When the fraction collector is full of eluted oligonucleotides, they aretransferred (e.g., by automated robotics or by hand) to a dryingstation. For example, in some embodiments, the samples are transferredto customized racks for Genevac centrifugal evaporator to be dried down.In preferred embodiments, the Genevac evaporator is equipped with racksdesigned to be used in both the Genevac and the subsequent desaltingstep. The Genevac evaporator decreases drying time, relative to othercommercially available evaporators, by 60%.

D. Desalting Component

In some embodiments, following HPLC, oligonucleotides are desalted. Inother embodiments, oligonucleotides are not HPLC purified, but insteadproceed directly from deprotection to desalting. In some embodiments,the desalting stations have TECAN robot systems for automated desalting.The system employs a rack that has been designed to fit the TECAN robotand the Genevac centrifugal evaporator without transfer to a differentrack or holder. The racks are designed to hold the different sizes ofdesalting columns, such as the NAP-5 and NAP-10 columns. The TECAN robotloads each oligonucleotide onto an individual NAP-5 or NAP-10 column,supplies the buffer, and collects the eluate. If desired, desaltedoligonucleotides may be frozen or dried down at this point.

In some embodiments, following desalting, INVADER and targetoligonucleotides are analyzed by mass spectroscopy. For example, in someembodiments, a small sample from the desalted oligonucleotide sample isremoved (e.g., by a TECAN robot) and spotted on an analysis plate, whichis then placed into a mass spectrometer. The results are analyzed andprocessed by a software routine. Following the analysis, failedoligonucleotides are automatically reordered, while oligonucleotidesthat pass the analysis are transported to the next processing step. Thispreliminary quality control analysis removes failed oligonucleotidesearlier in the processing, thus resulting in cost savings and improvingcycle times.

E. Oligonucleotide Dilution and Fill Component

In some embodiments, the oligonucleotide production process furtherincludes a dilute and fill module. In some embodiments, each moduleconsists of three automated oligonucleotide dilution and normalizationstations. Each station consists of a network-linked computer and anautomated robotic system (e.g., including but not limited to Biomek2000). In one embodiment, the pipetting station is physically integratedwith a spectrophotometer to allow machine handling of every step in theprocess. All manipulations are carried out in a HEPA-filteredenvironment. Dissolved oligonucleotides are loaded onto the Biomek 2000deck the sequence files are transferred into the Biomek 2000. The Biomek2000 automatically transfers a sample of each oligonucleotide to anoptical plate, which the spectrophotometer reads to measure the A260absorbance. Once the A260 has been determined, an Excel programintegrated with the Biomek software uses absorbance and the sequenceinformation to prepare a dilution table for each oligonucleotide. TheBiomek employs that dilution table to dilute each oligonucleotideappropriately. The instrument then dispenses oligonucleotides into anappropriate vessel (e.g., 1.5 ml microtubes).

In some preferred embodiments, the automated dilution and fill system isable to dilute different components of a kit (e.g., INVADER and probeoligonucleotides) to different concentrations. In other preferredembodiments, the automated dilution and fill module is able to dilutedifferent components to different concentrations specified by the enduser.

F. Quality Control Component

In some embodiments, oligonucleotides undergo a quality control assaybefore distribution to the user. The specific quality control assaychosen depends on the final use of the oligonucleotides. For example, ifthe oligonucleotides are to be used in an INVADER SNP detection assay,they are tested in the assay before distribution.

In some embodiments, each SNP set is tested in a quality control assayutilizing the Beckman Coulter SAGIAN CORE System. In some embodiments,the results are read on a real-time instrument (e.g., a ABI 7700fluorescence reader). The QC assay uses two no target blanks as negativecontrols and five untyped genomic samples as targets. For consistency,every SNP set is tested with the same genomic samples. In preferredembodiment, the ADS system is responsible for tracking tubes through theQC module. Thus, in some embodiments, if a tube is missing, the ADSprogram discards, reorders, or searches for the missing tube.

In some preferred embodiments, the user chooses which QC method to run.The operator then chooses how many sets are needed. Then, in someembodiments, the application auto-selects the correct number of SNPsbased on priority and prints output (picklist). If a picklist needs tobe regenerated, the operator inputs which picklist they are replacing aswell as which sets are not valid. The system auto-selects the valid SNPsplus replacement SNPs and print output. Additionally, in someembodiments, picklists are manually generated by SNP number.

The auto-selected SNPs are then removed from being listed as availablefor auto-selection. In some embodiments, the software prints thefollowing items: SNP/Oligo list (picklist), SNP/Oligo layout (racksetup). The operator then takes the picklist into inventory and removesthe completed oligonucleotide sets. In some embodiments, a completed setis unavailable. In this case, the operator regenerates a picklist. Then,in preferred embodiments, the missing SNP set or tube is flagged in thesystem. Once a picklist is full, the oligonucleotides are moved to thenext step.

In some embodiments, the operator then takes the rack setup generated bythe picklist and loads the rack. Alternatively, a robotic handlingsystem loads the rack. In preferred embodiments, tubes are scanned asthey are placed onto the rack. The scan checks to make sure it is thecorrect tube and displays the location in the rack where the tube is tobe placed.

Completed racks are then placed in a holding area to await the robotprep and robot run. Then, in some embodiments, the operator views whatracks are in the queue and determines what genomics and reagent stockwill be loaded onto the robot. The robot is then programmed to perform aspecific method. Additionally, in some embodiments, the robot oroperator records genomics and reagents lot numbers.

In preferred embodiments, a carousel location map is printed thatoutlines where racks are to be placed. The operator then loads the robotcarousel according to the method layout. The rack is scanned (e.g., bythe operator or by the ADS program). If the rack is not valid for thecurrent robot method, the operator will be informed. The carousellocation for the rack is then displayed. The output plates are thenscanned (e.g., by the operator or by the ADS program). If the plate isnot valid for the current method the operator is informed. The carousellocation for the plate is then displayed.

Then, in some embodiments, the robot is run. The robot then places theplates onto heatblocks for a period of time specified in the method. Insome embodiments, the robot then scans the plates on the Cytofluor.Output from the cytofluor is read into the database and attached to theoutput plate record.

In other embodiments, the output is read on the ABI 7700 real timeinstrument. In some embodiments, the operator loads the plate on to the7700. Alternatively, in other embodiments, the robot loads the plateonto the ABI 7700. A scan is then started using the 7700 software. Whenthe scan is completed the output file is saved onto a computer harddrive. The operator then starts the application and scans in the platebar code. The software instructs the user to browse to the saved outputfile. The software then reads the file into the database and deletes thefile (or tells the operator to delete the file).

The plate reader results (e.g., from a Cytofluor or a ABI 7700) are thenanalyzed (e.g., by a software program or by the operator). Additionally,in some embodiments, the operator reviews the results of the softwareanalysis of each SNP and takes one of several actions. In someembodiments, the operator approves all automated actions. In otherembodiments, the operator reviews and approves individual actions. Insome embodiments, the operator marks actions as needing additionalreview. Alternatively, in other embodiments, the operator passes onreviewing anything. Additionally, in some embodiments, the operatoroverrides all automated actions.

Depending on the results of the QC analysis, one of several actions isnext taken. If the software marks ready for Full Fill, the operatorforwards discards diluted Probe/INVADER oligonucleotide mixes andforwards the samples to the packaging module.

If an oligonucleotide set fails quality control, the data is interpretedto determine the cause of the failure. The course of action isdetermined by such data interpretation. If the software marks anoligonucleotide Reassess Failed Oligonucleotide, no action by user isrequired, the reassess is handled by automation. In the software marksan oligonucleotide Redilute Failed Oligonucleotide, the operatordiscards diluted tubes. No other action is required. If the softwaremarks an oligonucleotide Order Target Oligonucleotide, no action by useris required. In this case, a synthetic target oligonucleotide is orderedfor further testing. If the software marks an oligonucleotide FailOligo(s) Discard Oligo(s), the operator discards the diluted tubes andun-diluted tubes. No other action is required. If the software marks anoligonucleotide Fail SNP, the operator discards the diluted andun-diluted tubes. No other action is required. If the software marks anoligonucleotide Full SNP Redesign, the operator discards the diluted andun-diluted tubes. No other action is required. If the software marks anoligonucleotide Partial SNP Redesign the operator discards diluted tubesand discards some un-diluted tubes. No other action is required.

In some embodiments, the software marks an oligonucleotide ManualIntervention. This step occurs if the operator or software hasdetermined the SNP requires manual attention. This step puts the SNP “onhold” in the tracking system while the operator investigates the sourceof the failure.

When a set of oligonucleotides (e.g., a INVADER assay set) is completed,the set is transferred to the packaging station.

4. Packaging Component

In some embodiments, one or more components generated using the systemof the present invention are packaged using any suitable means. In someembodiments, the packaging system is automated. In some embodiments, thepackaging component is controlled by the centralized control network ofthe present invention.

5. Centralized Control Network

In some embodiments, the automated DNA production process furthercomprises a centralized control system. In some embodiments, thecentralized control system comprises a computer system.

In some embodiments, the computer system comprises computer memory or acomputer memory device and a computer processor. In some embodiments,the computer memory (or computer memory device) and computer processorare part of the same computer. In other embodiments, the computer memorydevice or computer memory are located on one computer and the computerprocessor is located on a different computer. In some embodiments, thecomputer memory is connected to the computer processor through theInternet or World Wide Web. In some embodiments, the computer memory ison a computer readable medium (e.g., floppy disk, hard disk, compactdisk, DVD, etc). In other embodiments, the computer memory (or computermemory device) and computer processor are connected via a local networkor intranet. In certain embodiments, the computer system comprises acomputer memory device, a computer processor, an interactive device(e.g., keyboard, mouse, voice recognition system), and a display system(e.g., monitor, speaker system, etc.).

In preferred embodiments, the systems and methods of the presentinvention comprise a centralized control system, wherein the centralizedcontrol system comprises a computer tracking system (tracking software).As discussed above, the items to be manufactured (e.g. oligonucleotideprobes, targets, etc) are subjected to a number of processing steps(e.g. synthesis, purification, quality control, etc). Also as discussedabove, various components of a single order (e.g. one type of SNPdetection kit) are manufactured in separate tubes, and may be subjectedto a different number of processing steps. Consequently, the presentinvention provides systems and methods for tracking the location andstatus of the items to be manufactured such that multiple components ofa single order can be separately manufactured and brought back togetherat the appropriate time. The tracking system and methods of the presentinvention also allow for increased quality control and productionefficiency.

In some embodiments, the computer tracking system comprises a centralprocessing unit (CPU) and a central database. The central database isthe central repository of information about manufacturing orders thatare received (e.g. SNP sequence to be detected, final dilutionrequirements, etc), as well as manufacturing orders that have beenprocessed (e.g. processed by software applications that determineoptimal nucleic acid sequences, and applications that assign uniqueidentifiers to orders). Manufacturing orders that have been processedmay generate, for example, the number and types of oligonucleotides thatneed to be manufactured (e.g. probe, INVADER oligonucleotide, synthetictarget), and the unique identifier associated with the entire order aswell as unique identifiers for each component of an order (e.g. probe,INVADER oligonucleotide, etc). In certain embodiments, the components ofan order proceed through the manufacturing process in containers thathave been labeled with unique identifiers (e.g. bar coded test tubes,color coded test tubes, etc.).

In certain embodiments, the computer tracking system further comprisesone or more scanning units capable of reading the unique identifierassociated with each labeled container. In some embodiments, thescanning units are portable (e.g. hand held scanner employed by anoperator to scan a labeled container). In other embodiments, thescanning units are stationary (e.g. built into each module). In someembodiments, at least one scanning unit is portable and at least onescanning unit is stationary (e.g. hand held human implemented device).

Stationary scanning units may, for example, collect information from theunique identifier on a labeled container (i.e. the labeled container is‘red’) as it passes through part of one of the production modules. Forexample, a rack of 100 labeled containers may pass from the purificationmodule to the dilute and fill module on a conveyor belt or othertransport means, and the 100 labeled containers may be read by thestationary scanning unit. Likewise, a portable scanning unit may beemployed to collect the information from the labeled containers as theypass from one production module to the next, or at different pointswithin a production module. The scanning units may also be employed, forexample, to determine the identity of a labeled container that has beentested (e.g. concentration of sample inside container is tested and theidentity of the container is determined).

The scanning units are capable of transmitting the information theycollect from the labeled containers to a central database. The scanningunits may be linked to a central database via wires, or the informationmay be transmitted to the central database. The central databasecollects and processes this information such that the location andstatus of individual orders and components of orders can be tracked(e.g. information about when the order is likely to complete themanufacturing process may be obtained from the system). The centraldatabase also collects information from any type of sample analysisperformed within each module (e.g. concentration measurements madeduring dilute and fill module). This sample analysis is correlated withthe unique identifiers on each labeled container such that the status ofeach labeled container is determined. This allows labeled containersthat are unsatisfactory to be removed from the production process (e.g.information from the central database is communicated to robotic orhuman container handlers to remove the unsatisfactory sample). Likewise,containers that are automatically removed from the production process asunsatisfactory may be identified, and this information communicated to acentral database (e.g. to update the status of an order, allow are-order to be generated, etc). Allowing unsatisfactory samples to beremoved prevents unnecessary manufacturing steps, and allows theproduction of a replacement to begin as early as possible.

As mentioned above, the tracking system of the present invention allowsthe production of single orders that have multiple components that mayproceed through different production modules, and/or that may beprocessed (at least in part) in separate containers. For example, anorder may be for the production of an INVADER assay detection kit. AnINVADER assay detection kit is composed of at least 2 components (theINVADER oligonucleotide, and the downstream probe), and generallyincludes a second downstream probe (e.g. for a different allele), andone or two synthetic targets so controls may be run (i.e. an INVADERassay kit may have 5 separate oligonucleotide sequences that need to begenerated). The generation of separate sequences, in separatecontainers, generally necessitates that the tracking system track thelocation and status of each container, and direct the proper associationof completed oligonucleotides into a single container or kit. Providingeach container with a unique identifier corresponding to a single typeof oligonucleotide (e.g. an INVADER oligonucleotide), and alsocorresponding to a single order (a SNP detection kit for diagnosing acertain SNP) allows separate, high through-put manufacture of thevarious components of a kit without confusion as to what componentsbelong with each kit.

Tracking the location and status of the components of a kit (e.g. a kitcomposed of 5 different oligonucleotides) has many advantages. Forexample, near the end of the purification module HPLC is employed, and asimple sample analysis may be employed on each sample in each containerto determine if a sample is collected in each tube. If no sample iscollected after HPLC is performed, the unique identifier on thecontainer, in connection with the central database, identifies the typeof sample that should have been produced (e.g. INVADER oligonucleotide)and a re-order is generated. Identification of this particularoligonucleotide allows the manufacturing process for thisoligonucleotide to start over from the beginning (e.g. this order getspriority status over other orders to begin the manufacturing processagain). Importantly, the other components of the order may continue themanufacturing process without being discarded as part of a defectiveorder (e.g. the manufacturing process may continue for theseoligonucleotides up to the point where the defective oligonucleotide isrequired). Likewise, additional manufacturing resources are not wastedon the defective component (i.e. additional reagents and time are notspent on this portion of the order in further manufacturing steps).

The unique identifier on each of the containers allows the variouscomponents of a given order to be grouped together at a step when thisis required (likewise, there is no need to group the components of anorder in the manufacturing process until it is required). For example,prior to the dilute and fill module, the various components of a singleorder may be grouped together such that the contents of the propercontainers are combined in the proper fashion in the dilute and fillmodule. This identification and grouping also allows re-orders to ‘find’the other components of a particular order. This type of grouping, forexample, allows the automated mixing, in the dilute and fill stage, ofthe first and second downstream probes with the INVADER oligonucleotide,all from the same order. This helps prevent human errors in readingcontainers and accidentally providing probes intended for one SNP beinglabeled as specific for a different SNP (i.e. this helps preventcomponents of different kits from being accidentally mixed together).The identification of individual containers not only allows for theproper grouping of the various components of a single order, but alsoallows for an order to be customized for a particular customer (e.g. acertain concentration or buffer employed in the second dilute and fillprocedure). Finally, containers with finished products in them (e.g.containers with probes, and containers with synthetic targets) need tobe associated with each other so they are properly assayed in thequality control module, and packaged together as a single kit(otherwise, quality control and/or a final end-user may find falsenegative and false positives when attempting to test/use the kit). Theability to track the individual containers allows the components of akit to be associated together by directing a robot or human operatorwhat tubes belong together. Consequently, final kits are produced withthe proper components. Therefore, the tracking systems and methods ofthe present invention allow high through-put production of kits withmany components, while assuring quality production.

6. Production in Practice

This Example describes the production of an INVADER assay kit for SNPdetection using the automated DNA production system of the presentinvention.

A. Oligonucleotide Design

The sequence of the SNP to be detected is first submitted through theautomated web-based user interface or through e-mail. The sequences arethen transferred to the INVADER CREATOR software. The software designsthe upstream INVADER oligonucleotide and downstream probeoligonucleotide. The sequences are returned to the user for inspection.At this point, the sequences are assigned a bar code and entered intothe automated tracking system. The bar codes of the probe and INVADERoligonucleotide are linked so that their synthesis, analysis, andpackaging can be coordinated.

B. Oligonucleotide Synthesis

Once the probe and INVADER oligonucleotide sequences have been designed,the sequences are transferred to the synthesis component. The bar codesare read and the sequences are logged into the synthesis module. Eachmodule consists of 14 MOSS EXPEDITE 16-channel DNA synthesizers (PEBiosystems, Foster City, Calif.), that prepare the primary probes, andtwo synthesizers, e.g., ABI 3948 48-Channel DNA synthesizers or ABI 3900instruments (Applied Biosystems, Foster City, Calif.), that prepare theINVADER oligonucleotides. Synthesizing a set of two primary and INVADERprobes is complete 3-4 hours. The instruments run 24 h/day. Followingsynthesis, the automating tracking system reads the bar codes and logsthe oligonucleotides as having completed the synthesis module.

The synthesis room is equipped with centralized reagent delivery.Acetonitrile is supplied to the synthesizers through stainless steeltubing. De-blocking solution (e.g., 3% TCA in methylene chloride or 2%DCA in toluene) is supplied through Teflon tubing. Tubing is designed toattach to the synthesizers without any modification of the synthesizers.The synthesis room is also equipped with an automated waste removalsystem. Waste containers are equipped with ventilation and containsensors that trigger removal of waste through centralized tubing whenthe cache pots are full. Waste is piped to a centralized storagefacility equipped with a blow out wall. The pressure in the synthesisinstruments is controlled with argon supplied through a centralizedsystem. The argon delivery system includes local tanks supplied from acentralized storage tank.

During synthesis, the efficiency of each step of the reaction ismonitored. If an oligonucleotide fails the synthesis process, it isre-synthesized. The bar coding system scans the container of theoligonucleotide and marks it as being sent back for re-synthesis.

Following synthesis, the oligonucleotides are transported to thecleavage and deprotection station. At this stage, completedoligonucleotides are subjected to a final deprotection step and arecleaved from the solid support used for synthesis. The cleavage anddeprotection may be performed manually or through automated robotics.The oligonucleotides are cleaved from the solid support used forsynthesis by incubation with concentrated NaOH and collected. In someembodiments, the deprotection step takes about 8-12 hours. In otherembodiments, “fast deprotection” chemistry comprising use of amiditeshaving the tert.-butylphenoxy-acetyl “tac” base protecting group is used(Proligo, LLC., Boulder, Colo.). This protecting group decreasescleavage and deprotection time of the final oligo from to 15 minutes at55° C., or two hours at room temperature. Following cleavage, the barcode scanner scans the oligonucleotide tubes and logs them as havingcompleted the cleavage and deprotection step.

C. Purification

Following synthesis and cleavage, probe oligonucleotides are furtherpurified using HPLC. INVADER oligonucleotides are not purified, butinstead proceed directly to desalting (see below).

HPLC is performed on instruments integrated into banks (modules) of 8.Each HPLC module consists of a Leap Technologies 8-port injectorconnected to 8 automated Beckman-Coulter HPLC instruments. The automaticLeap injector can handle four 96-well plates of cleaved and deprotectedprimary probes at a time. The Leap injector automatically loads a sampleonto each of the 8 HPLCs.

Buffers for HPLC purification are produced by the automated bufferpreparation system. The buffer prep system is in a general access area.Prepared buffer is then piped through the wall in to clean room (HEPAenvironment). The system includes large vat carboys that receivepremeasured reagents and water for centralized buffer preparation. Thebuffers are piped from central prep to HPLCs. The conductivity of thesolution in the circulation loop is monitored as a means of verifyingboth correct content and adequate mixing. The circulation lines arefitted with venturis for static mixing of the solutions; additionalmixing occurs as solutions are circulated through the piping loop. Thecirculation lines are fitted with 0.05 mm filters for sterilization andremoval of any residual particulates.

Each purified probe is collected into a 50-ml conical tube in a carryingcase in the fraction collector. Collection is based on a set method,which is triggered by an absorbance rate change within a predeterminedtime window. The HPLC is run at a flow rate of 5-7.5 ml/min (the maximumrate of the pumps is 10 ml/min.) and each column is automatically washedbefore the injector loads the next sample. The gradient used isdescribed in Tables 3 and 4 and takes 34 minutes to complete (includingwash steps to prepare the column for the next sample). When the fractioncollector is full of eluted probes, the tubes are transferred manuallyto customized racks for concentration in a Genevac centrifugalevaporator. The Genevac racks, containing dry oligonucleotide, are thentransferred to the TECAN Nap10 column handler for desalting.

D. Desalting

Following HPLC purification (probe oligonucleotides) or cleavage(INVADER oligonucleotides), oligonucleotides move to the desaltingstation. The dried oligonucleotides are resuspended in a small volume ofwater. Desalting steps are performed by a TECAN robot system. The racksused in Genevac centrifugation are also used in the desalting step,eliminating the need for transfer of tubes at this step. The racks arealso designed to hold the different sizes of desalting columns, such asthe NAP-5 and NAP-10 columns. The TECAN robot loads each oligonucleotideonto an individual NAP-5 or NAP-10 column, supplies the buffer, andcollects the eluate.

E. Dilution

Following desalting, the oligonucleotides are transferred to the diluteand fill module for concentration normalization and dispenation. Eachmodule consists of three automated probe dilution and normalizationstations. Each station consists of a network-linked computer and aBiomek 2000 interfaced with a SPECTRAMAX spectrophotometer Model 190 orPLUS 384 (Molecular Devices Corp., Sunnyvale Calif.) in a HEPA-filteredenvironment.

The probe and INVADER oligonucleotides are transferred onto the Biomek2000 deck and the sequence files are downloaded into the Biomek 2000.The Biomek 2000 automatically transfers a sample of each oligonucleotideto an optical plate, which the spectrophotometer reads to measure theA260 absorbance. Once the A260 has been determined, an Excel programintegrated with the Biomek software uses the measured absorbance and thesequence information to calculate the concentration of eacholigonucleotide. The software then prepares a dilution table for eacholigonucleotide. The probe and INVADER oligonucleotide are each dilutedby the Biomek to a concentration appropriate for their intended use. Theinstrument then combines and dispenses the probe and INVADERoligonucleotides into 1.5 ml microtubes for each SNP set. The completedset of oligonucleotides contains enough material for 5,000 SNP assays.

If an oligonucleotide fails the dilution step, it is first re-diluted.If it again fails dilution, the oligonucleotide is re-purified orreturned for re-synthesis. The progress of the oligonucleotide throughthe dilution module is tracked by the bar coding system.Oligonucleotides that pass the dilution module are scanned as havingcompleted dilution and are moved to the next module.

F. Quality Control

Before shipping, the SNP set is subjected to a quality control assay ina SAGIAN CORE System (Beckman Coulter), which is read on a ABI 7700 realtime fluorescence reader (PE Biosystems). The QC assay uses two notarget blanks as negative controls and five untyped genomic samples astargets.

The quality control assay is performed in segments. In each segment, theoperator or automated system performs the following steps: log on;select location; step specific activity; and log off. The ADS system isresponsible for tracking tubes. If a tube is missing, existing ADSprogram routines will be used to discard/reorder/search for the tube.

In the first step, a picklist is generated. The list includes theidentity of the SNPs that are being tested and the QC method chosen. Thetubes containing the oligonucleotide are selected by the automatedsoftware and a copy of the picklist is printed. The tubes are removedfrom inventory by the operator and scanned with the bar code reader andbeing removed from inventory.

The operator or the automated system then takes the rack setup generatedby the picklist and loads the rack. Tubes are scanned as they are placedonto the rack. The scan checks to make sure it is the correct tube anddisplays the location in the rack where the tube is to be placed.Completed racks are placed in a holding area to await the robot prep androbot run.

The operator or the automated system then chooses the genomics andreagent stock to be loaded onto the robot. The robot is programmed withthe specific method for the SNP set generated. Lot numbers of thegenomics and reagents are recorded. Racks are placed in the propercarousel location. After all the carousel locations have been loaded therobot is run.

Places are then incubated on the robot. The plates are placed ontoheatblocks for a period of time specified in the method. The operatorthen takes the plate and loads it into the ABI 7700. A scan is startedusing the 7700 software. When the scan is completed the operatortransfers the output file onto a Macintosh computer hard drive. Theoperator then starts the analysis application and scans in the plate barcode. The software instructs the operator to browse to the saved outputfile. The software then reads the file into the database and deletes thefile.

The results of the QC assay are then analyzed. The operator scans platein at workstation PC and reviews automated analysis. The automatedactions are performed using a spreadsheet system. The automatedspreadsheet program returns one of the following results:

1) Mark SNP Oligonucleotide ready for full fill (Operator discardsdiluted Probe/INVADER mixes. Requires no other action).2) ReAssess Failed Oligonucleotide (Requires no action by operator,handled by automation).3) Redilute Failed Oligonucleotide (Operator discards diluted tubes.Requires no other action).4) Order Target Oligonucleotide (Requires no action by operator, handledby automation).5) Fail Oligo(s) Discard Oligo(s) (Operator discards diluted tubes.Operator discards un-diluted tubes. Requires no other action).6) Fail SNP (Operator discards diluted tubes. Operator discardsun-diluted tubes. Requires no other action).7) Full SNP Redesign (Operator discards diluted tubes. Operator discardsun-diluted tubes. Requires no other action).8) Partial SNP Redesign (Operator discards diluted tubes. Operatordiscards some un-diluted tubes. Requires no other action).9) Manual Intervention (This step occurs if the operator or software hasdetermined the SNP requires manual attention. This step puts the SNP “onhold” in the tracking system).

The operator then views each SNP analysis and either approves allautomated actions, approves individual actions, marks actions as needingadditional review, passes on reviewing anything, or over rides automatedactions. Once the SNP set has passed the QC analysis, theoligonucleotides are transferred to the packaging station.

The present invention has been described in terms of specificembodiments incorporating details to facilitate the understanding of theprinciples of construction and operation of the invention. Suchreference herein to specific embodiments and details thereof is notintended to limit the scope of the claims appended hereto. It will beapparent to those skilled in the art that modifications may be made inthe embodiment chosen for illustration without departing from the spiritand scope of the invention.

Improvement of DNA Synthesizer

The present invention provides means of modifying existingoligonucleotide synthesis instruments to improve efficiency,reliability, and safety. In these Examples, commercially availableinstruments are modified to provide improved synthesizers of the presentinvention.

EXAMPLE 1 The Northwest Engineering 48-Column OligonucleotideSynthesizer

The Northwest Engineering 48-Column Oligonucleotide Synthesizer (NEI-48,Northwest Engineering, Inc., Alameda, Calif.) is an “open system”synthesizer in that the dispensing tubes for the delivery of reagentsare not affixed to each synthesis vial or column for the entire term ofthe synthesis process. Instead, movement of a round cartridge containingthe columns allows each dispensing tube to serve multiple columns. Inaddition, when a synthesis column is positioned to receive reagent, thedispenser is not even temporarily affixed to the vial with a sealedcoupling. The reagent dispensed to the vial has open contact with thesurrounding environment of the chamber. The chamber containing thesynthesis vials is isolated from the ambient environment by a top plate.The general design and operation of the NEI instrument is described inWO 99/656602.

The NEI-48 synthesizer includes external mounting points for variousreagent bottles, such as the phosphoramidite monomers used to form thepolymer chain, and the oxidizers, capping reagents and deblockingreagents used in the reaction steps. TEFLON tubing feeds liquid fromeach reagent bottle to its assigned valve on the top of the machine. Thefeeding is done under pressure from an argon gas source.

The operations of the machine are controlled using a computer. Thecomputer is fitted with a motion control card connected via cabling to amotor controller in the synthesizer; in addition, the computer isconnected to the synthesizer via an RS-232C cable. The provided softwareallows the user to monitor and control the machine's synthesisoperations.

The machine also requires connection to a source of argon gas, to bedelivered at a pressure between 15 and 60 psi, inclusive, and a sourceof compressed air or nitrogen, to be delivered at a pressure between 60and 120 psi, inclusive.

Synthesis in the NEI-48 occurs within synthesizer columns that arearranged in the cartridge.

Operations of the NEI-48 in accordance with the manufacturer'sinstructions produced undesirable emissions and leakage resulting inpotential synthesis and instrument failure. The following sectiondetails two of the sources of these emissions, and details one or moreaspects of the present invention applied to solve each problem, tothereby improve the performance of this machine.

A. Column Overflow Due to Inadequate Argon Pressure

Undesirable emissions and exposure are increased when columns overflow,causing the hazardous reagents used during synthesis to collect in thechamber bowl. A number of types of malfunction in the machine can leadsto incomplete drainage or purge of the columns, and each will eventuallylead to column overflow as the instrument proceeds through itssubsequent dispensing steps.

The flow of reagent and waste from the synthesis columns is controlledby a differential in the pressure of argon between the top and bottomopenings of the column. When the pressure of argon on the top opening isnot sufficiently high, the column will not drain or be purgedcompletely, i.e., fluid that should be drained will remain in thecolumn. This improper purging not only reduces the efficiency of thesynthesis chemistry, it also leads to column overflow. Therefore,failure of either initial pressurization of the chamber, or leakage ofargon from any coupling (in an amount great enough to reduce either theoverall pressure of the system or the pressure differential across thesynthesis column) may lead to undesirable emissions and exposure. Oneaspect of the present invention is to prevent column overflow byreducing leakage of argon at a variety of points in the system.

The NEI-48 demonstrated a variety of failures as a result of argonleakage from or within the instrument. To address this problem, thedrain plate gasket 43 of the present invention was created and wasfitted between the cartridge and drain plate. Addition of the gasket tothis assembly, as diagramed in FIG. 6, provided a pressure-tight seal,thereby containing the argon and allowing proper drainage of the columnsat the purging step. The gasket of the present invention applied in thisway improved the safety of the machine, and improved the efficiency ofthe synthesis reaction.

In another embodiment, a modified drain plate gasket was provided. Thedrain plate has securing holes 33, for attachment of the motor connector22. The first gasket was of a design that avoided the areas of the motorconnector 22 and the securing holes 33. A modified drain plate gasketwas designed with guide holes 44 to fit closely around each securinghole 33, such that the holes served to place the gasket in a specificposition between the cartridge and the drain plate (FIG. 6). In analternative embodiment, the drain plate 19 and the cartridge 3 may beprovided with other alignment features, such as pin fittings andcorresponding pin receiving holes (not shown) to facilitate alignment ofthese parts during assembly (e.g., after cleaning). A modified drainplate gasket for use with these parts may be provided with pin guideholes (not shown). Use of either the securing holes 33, or pins fittingsto align the gasket makes the gasket easier to position during assembly,ensuring proper operation of the gasket and improving ease of anymaintenance that requires disassembly of these parts.

B. Emissions from Reagent Bottles

During normal operations and without any malfunction, fumes cannonetheless be emitted by the reagent bottles attached to the machine.These emissions can be increased by poor fit or incorrect seals aroundbottle caps. For example, the reagent bottles for the NEI-48 are affixedto the machine by clamps that apply pressure to the outside of thebottle caps. The clamps can distort the caps, increasing leakage andgaseous emissions.

One aspect of the present invention is to provide a means of collectingemissions from reagent bottles. For improving the NEI-48, a reagentstand comprising a ventilation tube was constructed. The stand holds thereagent bottles, thereby eliminating the need for the cap-distortingclamps, and consequently reducing emissions from the bottles; theventilation tube removes any remaining emitted gases. This reagentdispensing station improves the safety of the machine in normaloperation. The reagent dispensing station of the present invention isnot limited to a configuration comprising a stand. It is envisioned thata station comprising a ventilation system may also be used with one ormore bottles held in clamps. In preferred embodiments, at least oneaspect of the reagent container system, e.g., the clamp, the cap, or thebottle, is modified such that clamping the reagent bottle does notcompromise the containment function of the cap, or of any other aspectof the reagent container system.

EXAMPLE 2 The Applied Biosystems 3900 Oligonucleotide Synthesizer

The Applied Biosystems 3900 Oligonucleotide Synthesizer (AppliedBiosystems, Foster City, Calif.) is similar in design and function tothe NEI-48, described above. The 3900 is an “open system” synthesizerutilizing a round cartridge containing the columns. The receiving holesof the cartridge are essentially cylindrical, and, as with the NEI-48,proper function of the instrument relies on an airtight seal between thecolumns and cartridge.

The 3900 synthesizer includes recessed areas for the external mountingof reagent bottles. When mounted on the instrument, the reagent bottlesdo not protrude beyond the outside edges of the instrument; they arecompletely recessed, (as, e.g., the reagent reservoirs 72 are recessedin base 2, diagrammed in FIG. 13A). As with the NEI-48, the reagentfeeding is done under pressure from an argon gas source.

The performance of the 3900 synthesizer is improved using themodifications provided by the present invention. Two specificimprovements are described below. These particular improvements aredescribed by way of example; improvements to the ABI 3900 synthesizer,or any synthesizer, are not limited to the improvements described hereinbelow.

A. Column Overflow Due to Inadequate Argon Pressure

As described above for the NEI-48, the proper purging of the synthesiscolumns at each cycle relies on the maintenance of a differential inargon pressure between the top and bottom openings of the columns.Improper or incomplete purging reduces the efficiency of the synthesisand increases the risk of column overflow. Proper purging in the 3900,like other open systems, depends in part upon the formation of anairtight seal between receiving holes in the cartridge and exteriorsurfaces of the synthesis columns. The presence of irregularities in thecolumn shape or surface can prevent the formation of an airtight seal,allowing argon to leak around the column exterior, thereby disruptingthe pressure differential required to properly purge the columns at eachcycle. The need to discard columns having even minor imperfections addsexpense to the use of the instrument. If undetected, a faulty seal canlead to poor synthesis and column overflow, as described above.

As discussed above, in some embodiments, the present invention providesimproved synthesizers having reliable seals between the cartridge andthe synthesis columns. The present invention provides a number ofembodiments of synthesizers having such seals. For example, as describedabove, a synthesizer may be improved by the addition of a resilientseal, such as an O-ring, in the receiving hole of each cartridge.

To make this improvement, the 3900 is fitted with such O-rings forsafer, more reliable and more efficient performance. Examples of severalmeans of creating an improved seal between the outer surface of a column61 and a receiving hole 11 are diagrammed in FIGS. 12A-12C. While any ofthe embodiments of seals disclosed herein may be applied to the 3900instrument, in a preferred embodiment, the 3900 is improved by the useof an embodiment similar to that diagrammed in FIG. 12B, wherein agroove 70 creates a groove lip 71, to accommodate and hold an O-ring 67,thus providing a seal between cartridge 3 and the exterior surface 61 ofthe synthesis column 12. In a particularly preferred embodiment, thereceiving hole 11 is enlarged in diameter to facilitate insertion andremoval of an O-ring 67, e.g., for easy cleaning or replacement. Agroove is machined into the interior of each receiving hole in a 3900cartridge, and appropriate O-ring seals are placed in the grooves. Asnoted above, the O-ring could be of any suitable material. Thusmodified, the cartridge of the 3900 has a greatly improved ability toaccommodate imperfections in the exteriors of synthesis columns, andthis improvement results in safer, and more efficient and reliableoperation of the instrument, with fewer costs associated with chemicalspill clean-up, instrument down-time, and the disposal of unusablesynthesis columns.

B. Emissions from Reagent Bottles

During normal operations and without any malfunction, fumes arenonetheless emitted by the reagent bottles attached to the 3900 machine.These emissions can be significant, even though gaskets are provided foruse in conjunction with the bottle caps.

As described above, the present invention provides a means of collectingemissions from reagent bottles. On the 3900, the reagent bottles areattached in recessed areas on the exterior in the base of the instrument(e.g., the reagent reservoirs 72 attached to the recessed areas in thebase 2, as illustrated in FIG. 13A). The emissions from this instrumentare reduced by modification to provide the enclosed reagent dispensingstation of the present invention. In modification of the 3900, therecessed areas are provided with panels to enclose the space, reducingthe release of hazardous vapors.

Reagent bottles or reservoirs need to accessible for changing orfilling, due, e.g., to consumption of reagents during synthesisoperations. In making the modification to the 3900, the panels added tothe instrument are moveable, to provide access to the reagent bottleswithin the enclosed space. In a simple configuration, panels providedfor the purpose of enclosing the space are attached by use of strips ofVELCRO fastener (e.g., adhesive backed strips), for easy mounting andremoval. For a sturdier attachment, the panels may be attached usinghard, removable fasteners, such as screws or bolts. In a particularlypreferred configuration, the panels are mounted in tracks, brackets orother suitable fittings that allow them to be moved or removed bysliding.

To monitor reagent bottles (e.g., to determine when changing or fillingis needed), it is preferred that the reagent reservoirs be accessiblefor visual inspection. In making the addition of panels to the 3900, thepanels are constructed such that the reagent bottles can be visuallyinspected without opening the enclosure. The panels provided areconstructed of transparent material. While glass may be used, inpreferred embodiments, for both safety and ease of handling a plastic isused with sufficient transparency to allow visual inspection of reagentbottles, and with sufficient resistance to the chemicals used insynthesis to avoid rapid or immediate decay or fogging, (as is oftenassociated with exposure of plastics to vapors of solvents to which theyare not resistant), when used in this application. Selection of plasticsfor appropriate chemical resistance is well known in the art, and tablesof chemical compatibility are generally readily available frommanufacturers.

The panels are provided with a ventilation port (e.g., ventilation port74, as diagrammed in FIG. 13B), for the removal vapors and fumes emittedby the reagent bottles. Such a ventilation port serves as an attachmentpoint for a ventilation tube to conduct fumes away from the instrument,e.g., into an exhaust system. Since the vapors from DNA synthesisreagents tend to be heavier than air, the ventilation port is placednear the bottom of the enclosure. Placement of the ventilation porttoward the rear is convenient for attachment to a larger exhaust system,minimizes or prevents interference by the ventilation tubing withoperator access to other parts of the instrument, and conducts the fumesaway from instrument operators.

To maximize efficacy of the ventilation system, an air inlet into theenclosure is provided. In applying the panels to the 3900, a clearancebetween the attached panels and the body of the instrument (e.g., theclearance 75 between the panel 73 and the base 2 diagrammed in FIG. 13B)provides the air inlet. The panel is positioned such that the principalair inlet is a clearance between the front edge of the panel (i.e., theedge closest to the front of the instrument) and the instrument base.Positioning of the inlet toward the front of the instrument, or on theopposite side of an enclosure from a ventilation port, maximizes theflow of air through the enclosure, providing the most efficient removalof vapors. The inward flow of air minimizes the possible escape ofhazardous vapors toward instrument operators. Thus modified, the 3900instrument is improved with respect to its emissions of hazardousvapors.

C. Emissions from the Chamber Bowl

During normal operations and without any malfunction, fumes arenonetheless emitted when the chamber bowl of the ABI 3900 is opened foraccess by the instrument operator (e.g., when the lid is opened toretrieve columns after synthesis is completed). These emissions can besignificant. The present invention provides a means of collectingemissions from the 3900 without the use of a separate fume hood. Thepresent invention comprises a synthesizer having an integratedventilation system to contain and remove vapor emissions. One aspect ofthe invention is to collect and remove vapors when the instrument isopen. Embodiments of integrated ventilation systems as applied to the3900 instrument are shown in FIGS. 19-22.

As shown in FIG. 19A, in one embodiment, the lid enclosure 102 ismodified to comprise a ventilation opening 105. The lid enclosure of the3900 comprises an outer window 101. In preferred embodiments, aventilation opening is placed in the center of the outer window 101 ofthe lid enclosure 105, so as to avoid blocking the operator's view ofinternal components, such as the synthesis columns, during operation.

As shown in the diagram of FIG. 21, the lid enclosure of the 3900instrument comprises an outer window 101 and an inner window 25. Thespace between the windows is open to the ambient environment through aventilation slot 100 near the lid enclosure hinge 106. The outer windowin an unmodified instrument allows visual inspection of operations andcomponents within the lid enclosure and within the chamber bowl 18 ofthe base 2. Reagent supply tubing passes through the inner window, butthe window is sealed around each tube so that the chamber will maintainappropriate pressure during operation. In the embodiment shown in FIGS.19, 20 and 21, the ventilation opening provides an aperture in the outerwindow.

In another embodiment, one or more ventilation openings may be providedin the base (e.g., 2) of the synthesizer, as diagrammed in FIG. 22. Inother embodiments, a synthesizer may comprise ventilation openings inboth a lid enclosure and a base.

Each ventilation opening is attached to ventilation tubing (e.g., 103)for attachment to an exhaust system. In some embodiments, a synthesizeris attached to an individual exhaust system. In other embodiments,multiple synthesizers are attached to a centralized exhaust system. In apreferred configuration, the access to the exhaust system is toward therear of the instrument, to minimize or prevent interference by theventilation tubing with operator access to the chamber bowl, and toconduct the fumes away from instrument operators.

Another aspect of the present invention is to provide a ventilatedworkspace around the chamber bowl having a negative air pressurerelative to the surrounding air pressure, such that the flow of air goesfrom the surrounding room into the ventilated workspace, and not in thereverse, during operation of the ventilation system. The ventilatedworkspace is designed to allow the instrument operator to reach into thespace (e.g., to remove the synthesis columns) without turning off theventilation system. Embodiments of a ventilated workspace are shown inFIG. 20 A-C. As shown in this embodiment, the ventilated workspace iscreated by providing side panels between the body of the synthesizer andthe lid enclosure, and a front panel. The presence of the panels reducesthe size of the opening through which ambient air can enter theventilated workspace. When the ventilation system is turned on (i.e.,when the connected ventilation tube is drawing air from the ventilationopening, the airflow in through the reduced opening prevents or reducesany outward flow of gaseous emissions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

1. A nucleic acid synthesizer comprising a plurality of synthesiscolumns and an energy input component that imparts energy to saidplurality of synthesis columns to increase nucleic acid synthesisreaction rate in said plurality of synthesis columns.
 2. The synthesizerof claim 1, wherein said energy input component comprises a heatingcomponent.
 3. The synthesizer of claim 8, wherein said heating componentprovides substantially uniform heat to said plurality of synthesiscolumns.
 4. The synthesizer of claim 1, wherein said energy inputcomponent provides heated reagent solutions to said plurality ofsynthesis columns.
 5. The synthesizer of claim 1, wherein said energyinput heats said plurality of synthesis columns in the range of about 20to about 60 degrees Celsius.
 6. The synthesizer of claim 2, wherein saidheating component comprises at least one member selected from the groupconsisting of a heating coil, a heat blanket, a resistance heater, aPeltier device, a magnetic induction device, a microwave device, aheated fluid and a heated gas.
 7. The synthesizer of claim 1, whereinsaid energy input component provides energy in the electromagneticspectrum.
 8. The synthesizer of claim 1, wherein said energy inputcomponent comprises an oscillating member.
 9. The synthesizer of claim1, wherein said energy input component provides a periodic energy input.10. The synthesizer of claim 1, wherein said energy input componentprovides a constant energy input.
 11. The synthesizer of claim 1,further comprising a mixing component that mixes reagents in saidplurality of synthesis columns.
 12. The synthesizer of claim 11, whereinsaid mixing component is selected from the group consisting of anultrasonic mixer, a magnetic mixer, a fluid oscillator, and avibrational mixer.
 13. The synthesizer of claim 1, further comprising areaction support, said reaction support configured to hold three or moresynthesis columns.
 14. The synthesizer of claim 13, wherein saidreaction support is configured for operation with a cleavage anddeprotect component.
 15. The synthesizer of claim 14, further comprisinga robotic component configured to transfer said reaction support fromsaid synthesizer to said cleavage and deprotect component.
 16. Thesynthesizer of claim 15, wherein said robotic component is furtherconfigured to transfer said reaction support from said cleavage anddeprotect component to a purification component.
 17. A nucleic acidsynthesizer comprising a plurality of synthesis columns and a mixingcomponent that mixes reagents in said plurality of synthesis columns.18. The nucleic acid synthesizer of claim 17, wherein said mixer isselected from the group consisting of an ultrasonic mixer, a magneticmixer, a fluid oscillator, and a vibrational mixer.
 19. The nucleic acidsynthesizer of claim 17, further comprising an energy input componentthat imparts energy to said plurality of synthesis columns to increasenucleic acid synthesis reaction rate in said plurality of synthesiscolumns.
 20. A nucleic acid synthesizer, comprising; a) a ventilationtube, and b) a lid enclosure comprising; a) a top cover with aventilation slot, and b) a top enclosure comprising a top plate with aventilation opening, wherein said top enclosure is attached to said topcover to form a primarily enclosed space over said top cover; and c) aplurality of synthesis columns; and d) an energy input component thatimparts energy to said plurality of synthesis columns to increasenucleic acid synthesis reaction rate in said plurality of synthesiscolumns.